716918 [Reprinted from RADIOLOGY, Vol. 39, No. 3, Pages 541-572, November, 1942 I Copyrighted 1942 by the Radiological Society of North America, Incorporated The Use of Radioactive Tracers in Biology and Medicine’ JOSEPH G.HAMILTON, M.D. Crocker Radiation Laboratory, University of California, Berkeley, Calif.

VERY LARGE share of investigative metabolism of the substances that are A work in the biological sciences has being studied. Second, it is not possible been devoted to the study of the metabo- by chemical procedures to differentiate lism of organisms ranging in complexity between the atoms or molecules of the from bacteria to man and embracing the administered material and tho2e that are more important members of the plant and already present in the tissues of the plant animal kingdoms. Until the discovery of or animal. For example, the increase in artificial radioactivity by Curie and Joliot excretion of phosphorus by an animal in 1934 (l),the only method for studying following the administration of this ele- the mechanisms whereby the various ele- ment is made up in part by the phos- ments and compounds essential for life phorus atoms already in the body, which are assimilated, distributed throughout were displaced by the administered ele- the tissues, converted into other com- ment, and in part by a portion of the pounds, and finally eliminated, was by a phosphorus given to the animal. The direct chemical approach to these prob- classical procedures of chemistry and lems. This mode of attack, although very physics do not permit the investigator productive of information, suffers from to determine what proportion of the phos- several serious limitations. First, in order phorus atoms in the excreta came from to observe the manner by which a living the administered material and the fraction organism metabolizes an element, as, for that was displaced from the pre-existing example, potassium, it is necessary to phosphorus in the tissues. This limitation administer enough so that a detectable applies to all metabolic investigations in increase in the amount in the body will be which the elements or compounds are produced. This axiom applies to most of constituents of the organism that is being the elements and compounds that are studied. normal constituents of biological systems. The first attempt to circumvent these Normally, the content of these substances limitations was made by Hevesy in 1923 is kept in most instances within relatively (2), when he employed a radioactive iso- narrow limits by the body and for this tope of lead (radium D) to investigate the reason it is necessary to administer a metabolism of that element in plants. sufficient quantity of the element or com- His procedure made it possible to measure pound to disturb this carefully regulated quantitatively the uptake and distribution balance if fluctuations are to be produced of the administered lead by determining that are large enough to be accurately de- the radioactivity of the different portions termined by analytical chemical technics. of the plant. Due to the fact that de- It is obvious that such procedures will fre- tection of radio-lead by its radioactivity quently disturb the normal chemical and is more than a million times more sensitive physiological processes of the organism, and than the ordinary chemical and physical the experimental data thus obtained may methods for its determination, it was not present a true picture of the normal possible to study the metabolism of this - element in such minute quantities that its From the Crocker Radiation Laboratory and the toxic effects were avoided. Later, similar Divisions of Medicine and Radiology of the University of California Medical School. Presented, as a part of a investigations were undertaken with radio- Symposium on the Cyclotron, before the Radiological bismuth (radium The applications of Society of North America, at the Twenty-seventh An- E). nual Meeting, San Francisco, Calif., Dec. 1-5, 1941. this technic were of limited value at that 542 JOSEPH G. HAMILTON November 1942

time, as lead and bismuth are not normal in tracer studies, since the amounts of constituents of biological systems. The radioactive material needed for this type discovery of artificial radioactivity by of investigation are far too minute to Curie and Joliot (1) and the development produce any chemical or physiological of the cyclotron by Lawrence and his changes by the action of their radiations. associates (3)have resulted in the prepara- Three general technics have been de- tion and identification of radioactive iso- veloped for the use of the radioactive iso- topes of all of the stable elements. These topes of stable elements as tracers in the momentous developments have given the biological sciences. First, the assimilation biologist probably the most useful tool of the administered radio-element, its for research since the discovery of the distribution in the tissues, its conversion microgcope, because almost all of the into other compounds by the body, and elements and compounds present in bio- finally its elimination may be followed logical systems can be “tagged” with the quantitatively by direct measurement of aid of artificial radio-elements and their the radioactivity of samples of tissues course in living structures directly studied. after their removal from the body. Sec- Chiewitz and Hevesy were the first in- ondly, the selective accumulation of the vestigators to employ the new tool of radioactive isotopes of several elements artificial radioactivity for biological re- may be observed in the organs and tissues search (4), and their initial studies were of the intact animal by measurement of devoted to the investigation of the metabo- the radioactivity of these structures in situ. lism of phosphorus in rats, with the aid Thirdly, distribution of the accumulated of a radioactive isotope of that element. radio-element in tissues may be studied Although only seven years have elapsed with the aid of photographic films. since their first experiments with radio- The first technic is the most widely phosphorus, artificial radioactivity has employed at present and is applicable to been applied as a tool in practically all of all the radio-elements. The radioactive the biological sciences and the radioactive isotope is usually given in the form of a isotopes of twenty-one elements have simple inorganic compound. For example, already been employed in these various -- radio-sodium _usually is administered as fields of investigation as tracers for meta- iron, of phospholipids and nucleo- differs only in its property of radio- proteins from inorganic phosphates, and of activity ; the chemical and physiological thyroxine from iodine-all have been properties of the two forms of the element studied with the aid of radioactive tracers. are identical so long as the radiations from In experiments of this type the compound the radioactive isotope are not sufficiently is isolated from the tissue and its radio- intense to produce physiological changes. activity is compared with the total amount This limiting factor is not of consequence of radio-element originally administered. Vol. 39 RADIOACTIVETRACERS IN BIOLOGY AND MEDICINE 543

Complex organic compounds may be artificial radio-elements, by means of labelled for tracer studies by the inclusion which the distribution of the administered of radioactive atoms in the molecules. labelled elements or compounds in tissues For example, thiamin (vitamin B,) has may be investigated. Thin sections of the been tagged by synthesizing it from radio- radioactive tissues are placed against sulfur. The labelled thiamin is then given photographic films. After an interval of to the animal and its fate in the body is time sufficient for adequate exposure, the followed by measuring the distribution film is removed and developed. The of the radioactivity from the radio-sulfur sections are stained, and each section with in the tissues, body fluids, and excreta. its corresponding piece of developed film By this procedure, the conversion by the (radio-autograph) is examined under the body of the labelled compound into other microscope. The areas of darkening in the compounds can be followed. film correspond to the regions of the The second general technic makes use tissues in which the greatest deposition of of the ability of many artificial radio- the radio-element has taken place. Thus elements to emit penetrating gamma rays a correlation between the deposition of the which pass through many centimeters of labelled element or compound and the tissue without significant absorption and histological structure of the tissue can be which can be measured at a considerable established. distance from the site of origin. The presence of the accumulated radio-element PHOSPHORUS in a particular organ is detected by placing The absorption of phosphorus in experi- P a suitable measuring device, such as a mental animals and in man has been ,Geiger counter tube, over it and measuring studied with the aid of radio-phosphorus the intensity of the gamma rays emitted by Hevesy and his co-workers (4, 5, 6), from the radioactive atoms which have Lawrence and his associates (7, 8), and been stored selectively in the tissue. This Cohn and Greenberg (9). These in- technic is illustrated in the study of the vestigators found that inorganic phos- iodine metabolism of the thyroid gland in phates were efficiently absorbed from the normal and goitrous human subjects. digestive tracts of both animal and man. The selective deposition of the admin- Usually less than 30 per cent of the ad- istered radio-iodine by the thyroid is ministered phosphate escaped assimilation determined by placing a Geiger counter when the labelled phosphorus was given tube over the neck and measuring the to subjects in the fasting state. The intensity of the gamma rays emitted from administration of either glucose or neutral the radio-iodine accumulated in the thy- fat with the phosphate slightly enhanced roid tissue. Since by this method the its absorption. The intravenous adminis- necessity of removing tissue for deter- tration to normal human subjects of di- mination of its radioactivity is eliminated, sodium phosphate which had been tagged it offers two distinct advantages. First, with radio-phosphorus was followed by it enables the observer to follow the ebb the elimination of from 4 to 23 per cent and flow of the labelled element or com- of the labelled phosphorus during the pound in the same human subject or animal first twenty-four hours after the experi- for a considerable period of time. The ments. Thereafter, the rate of excretion results thus obtained give a continuous fell rapidly and after the third day its record of the fate of the accumulated daily value was less than 1 per cent. The radioactive atoms or molecules. Secondly, kidneys were the chief channel of elimina- it makes possible in vivo tracer studies in tion, but the digestive tract accounted for normal human subjects. approximately 10 per cent of the tagged The third technic makes use of the phosphorus that left the body. When the photographic action of the radiations from labelled phosphorus was given by mouth 544 JOSEPH G. HAMILTON November 1942

to human subjects and to experimental that the turnover of phosphorus in the animals, from 10 to 40 per cent of the brain is much more gradual than that in adniinistered dose escaped assimilation the other tissues included in this series. and was eliminated in the feces. The These studies were repeated by Jones excretory pattern of the assimilated phos- et al. (10, ll), who compared the uptake phorus was essentially the same as that of of labelled phosphorus in normal animal the phosphorus administered by vein. tissues and in experimental tumors. The When the tagged phosphorus was given results of these experiments are sum- orally, however, approximately 25 per marized in the curves shown in Figure 1. cent of it was lost because of the failure of Significant information has been secured the body to assimilate it completely. from this study and from other experi- ments on normal and tumor tissue. Ac- cordingly, the degree of uptake of phos- phorus by each tissue apparently is de- pendent upon three factors: first, the total phosphorus content of the tissue; secondly, its rate of turnover in the tissue; thirdly, the laying down of new tissue. The relatively great accumulation of administered radio-phosphorus in bone, which is composed largely of calcium phos- phate, is an example of the first factor. The effect of the rate of turnover of phosphorus in tissue upon the uptake of a single dose of labelled phosphorus is clearly illustrated in the marked difference between the accumulation of radio-phos- phorus in liver and in brain. The total Fig. 1. Distribution of labelled phos- content of phosphorus in these two tissues phorus in normal and neoplastic tissues of is similar, while the uptake of labelled mice. phosphorus differs enormously. The rela- The distribution of phosphorus in animal tively low uptake in brain tissue indicates tissue has been extensively studied by the that it has a much slower rate of phos- workers listed in the preceding paragraph phorus metabolism than such tissue as as well as by many other investigators. liver, kidney, and small intestine. Since Cohn and Greenberg (9) observed that the in adult animals the total phosphorus retention of a single dose of di-sodium content of these tissues remains constant phosphate which had been tagged with within relatively narrow limits, the pres- radio-phosphorus varied in different tissues ence of a high proportion of labelled pe' unit of fresh weight in the following phosphorus in a tissue such as the liver decreasing order : bone, liver, stomach and indicates that the rate of turnover of phos- small intestine, heart, kidneys, lungs, phorus atoms between the cells of the liver blood, muscle, skin, and brain. All these and the blood stream is more rapid than tissues, with the exception of the brain, in brain tissue. In other words, an atom showed a rapid uptake during the first ten of phosphorus remains within a cortical hours after administration, followed by a neuron for a much longer interval of time prolonged and steadily diminishing loss. than in a hepatic cell. Many investigators In the brain a very gradual and prolonged have demonstrated that most of the uptake was followed by a very slow release labelled phosphorus taken into the different of the accumulated labelled phosphorus. tissues is bound in firm organic combina- This finding was interpreted to indicate tion Therefore, the varying rates of I lob350 1 ,i ,i

i Vol. 39 RADIOACTIVETRACERS IN BIOLOGY AND MEDICINE 545 phosphorus turnover apparently are a co-workers (15). These investigators dem- manifestation of the different speeds with onstrated that phospholipids are rapidly which the cells of the body build up and formed in the body from administered break down thc complex organic phos- inorganic phosphates, Artom and his phorus compounds that make up their group extracted the phospholipids from internal structure. The third factor which different organs of rats after the animals influences the degree of uptake of phos- had received radio-phosphorus, and found phorus is the rate of growth and develop- that the phospholipids from the liver, ment of new tissue. This phase of phos- kidneys, and small intestine had the I phorus metabolism is illustrated by the maximum radioactivity, that those from data on tumor tissue shown in Figure 1. the heart, lungs, spleen, pancreas, adre- Similar observations have been made on nals, and testes had medium values, and neoplasms in man (12). In these in- that those from muscle and brain had the stances the tumor cells constantly are lowest values. These observations were multiplying and laying down new tissue. interpreted to signify that the rates of , Another example of the effect of growth synthesis of phospholipids varied in the upon phosphorus metabolism is given in different organs. Chaikoff and his col- the study by Chaikoff and hisco-workers leagues (16-19) not only confirmed these (13) on the deposition of labelled phos- results in different animals but also demon- pholipids in the central nervous system of strated that synthesis of phospholipids rats at various stages of development. takes place in vitro within slices of excised The animals, ranging from newborn to liver, kidney, and small intestine. Since I fully developed, received a single dose of adult animals were used in these experi- labelled di-sodium phosphate and were ments, the quantities of labelled phos- sacrificed twenty-four and forty-eight pholipids found in the different tissues hours later. The different portions of the probably were an expression of the varying central nervous system were removed, and rates of replacement of phospholipids in the radio-phosphorus content of the ex- these structures. tracted phospholipids was determined for A comparison of the phosphorus metabo- each sample. The greatest deposition of lism of 'the tissues of normal and leu- radioactive phospholipids was found in the kemic mice was made by Lawrence and nerve tissue of the newborn animals. his associates (20, 21). They administered With increasing age of the animals, a a single dose of labelled phosphorus to precipitous decline in the labelled phos- each of a large group of normal and leu- pholipid content of the tissue occurred, kemic mice. The animals were sacrificed This observation indicates that the rate at varying intervals and the content of of synthesis and laying down of phos- both total and labelled phosphorus in the pholipids was greatest in the newborn tissues was determined. Figure 2 indi- animals and diminished as the tissues of cates the retention of radio-phosphorus per the central nervous system approached gram of tissue. It will be seen that there maturity. The three factors which in- is a striking difference between the accu- fluence the selective accumulation of radio- mulation of radio-phosphorus in normal phosphorus operate in a similar manner and in leukemic lymph nodes and spleens. when other elements are used. This The slight differencesapparent between the relationship will be apparent again in the uptake of the labelled phosphorus in the discussion of the selective uptake of other normal and the leukemic bone were inter- labelled elements in different tissues. preted as being due to the fact that the The conversion of labelled inorganic phosphorus metabolism of the marrow was i phosphorus into organic phosphorus com- masked by that of the bone. Normally I pounds was first studied by Hevesy and bone has a high content of phosphorus and his associates (14) and by Artom and his also the ability to accumulate relatively I

I

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I 546 JOSEPH G.HAMILTON November 1042

RETENTION OF PJ* PER GRAM IN VARIOUS TISSUES

MUSCLE I I I I I s- - 0 LCUNCYlO YlLl 0 LCUNCIK YKa s- 0 OOUTlOL 1101 - e CONTIOL YlCC 7- - I- - *t -I c 0- - :.- - 2,-

LIVER I I I I I 8- - 0 LCUNCYlO 1101 0 LCUlCYlO MlCI * -. 0 CONTROL YK. - 0 OO*lIOL YICC I- - s- -

I I 1 I I I I I 1 I

0

HOURS Fig. 2. Distribution of radio-phosphorus in the tissues of normal and leukemic mice.

large quantities of the administered A comparison of the rates of conversion labelled phosphorus. The total phos- of labelled inorganic phosphorus into phos- phorus content of the corresponding organs pholipids by normal tissues and by tumors in normal and leukemic animals did not in experimental animals was made by present any significant variations. The Jones, Chaikoff, and Lawrence (22, 23). greater accumulation of the labelled phos- They compared the rates of phospholipid phorus in the leukemic spleens and lymph synthesis in four different animal tumors nodes, although their total content was (carcinoma, lymphoma, sarcoma 180, and similar to that of the normal tissues, was lymphosarcoma) and found that each had a interpreted as an indication of the rapid characteristic pattern of phospholipid de- laying down of new tissue as well as of an position which was not related to cell type. increased metabolic activity of the pre- In each instance, however, the rate of existing leukemic cells. formation of the tagged phospholipids VOl. 39 RADIOACTIVETRACERS IN BIOLOGYAND MEDICINE

1 LIVER - LWKWG

SPLEEN - N I

a5

' 1234567

LYMPH-N

1234567

Fig. 3. Comparison of the rates of formation and distribution of the different organic phosphorus fractions in tumor tissue, and in normal and leukemic tissues. The ordinate indicates the per cent of the administered labelled di-sodium phosphate per gram of tissue which was converted into phospholipids, nucleoproteins, and the acid-soluble fraction. The abscissa represents the time in days following the administration of the radio-phosphorus. was rapid and in this respect resembled the that the characteristic rate of conversion phospholipid turnover of the more rapidly of radio-phosphorus into labelled phos- metabolizing tissues, such as the liver, pholipids was maintained for each type of kidney, and small intestine. In other tumor. They concluded that the phos- experiments these investigators implanted pholipid turnover of these three types of three different types of tumors (mammary tumor apparently remained independent carcinoma, lymphoma, and lymphosar- of the host. coma) into a large group of mice and found Later Tuttle et al. (24) compared the

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548 JOSEPH G. HAMILTON November 1942

also that the relative proportion of the administered phosphorus in the nuclei as compared to that in the cytoplasm was much greater in the malignant than in the normal cells (Fig. 4). The metabolic pattern of malignant cells was not re- stricted to neoplastic tissues but was found as well in rapidly multiplying normal cells. A comparison of the uptake of labelled phosphorus in the nuclei of malignant cells and of rapidly regenerating liver cells in animals from whom a large part of the liver had been removed revealed that the Fig. 4. Relative accumulation of radio-phos- phosphorus uptake by the nuclei of these phorus by the nuclei of liver cells from normal rats and the nuclei of tumor (lymphoma) cells in mice. two very different types of cells was essen- tially the same. From these observations the authors concluded that the variations distribution of labelled phosphorus in the in phosphorus metabolism of nuclei are acid-soluble phospholipid and nucleo- dependent] at least in part, upon the rate protein fractions of tissues of normal and of cell multiplication and are not a peculi- leukemic animals. The tissues were re- arity of malignancy fier se. It has been moved at varying intervals following the calculated from the observed rate of radio- administration of tagged di-sodium phos- phosphorus uptake by the nuclei of the phate. The different organic phosphorus lymphoma that an average of fifty-two fractions were extracted and their content hours was required for the formation of a of radio-phosphorus was determined. The new lymphoma nucleus, This value is in results of these studies are shown in part close agreement with the observed rate of in Figure 3. It will be seen that the growth of the tumor. Moreover, it has 1 nucleoprotein fraction from the tumor been calculated from these data that in the cells and from leukemic tissues contained lymphoma nucleus approximately 3 X lo4 considerably more radio-phosphorus than molecules of tetranucleotide were synthe- did the normal structures. These re- sized per second. The localization of sults were interpreted as indicating that radio-phosphorus in the nuclei of rapidly nucleoproteins were synthesized from the multiplying cells is of potential thera- assimilated inorganic phosphorus at a peutic significance, since this phenomenon more rapid rate by the leukemic and tumor suggests the possibility of the selective cells than by normal tissues. irradiation of that portion of the cell which The rBle of phosphorus in the metabo- plays a dominant rble in multiplication lism of normal and malignant tissues and other cellular functions. also has been demonstrated by Marshak The accumulation of labelled phosphorus (25, 26), who devised a technic for the in the tissues of patients suffering from separation of nuclei from cytoplasm. He different types of leukemia has been employed this procedure to determine the studied by Lawrence and his co-workers relative uptake of the administered in- (7, S, 12, 27, 28). In all these investiga- organic radio-phosphorus in the nuclei tions the radio-phosphorus was adminis- and cytoplasm of normal and malignant tered either orally or by vein in the form cells (lymphoma] sarcoma 180, and car- of di-sodium phosphate. They demon- cinoma 256). These studies revealed not strated that in human leukemia radio- only that the nuclei of the malignant cells phosphorus was selectively accumulated accumulated more of the labelled phos- in the leukocytes rather than in the eryth- phorus than those of the normal cells, but rocytes and plasma. The curves in Vol. 39 RADIOACTIVI~TRACERS IN BIOLOGYAND MEDICINE 549

Figure 5 indicate the variations in labelled phosphorus uptake in the components of leukemic blood. The distribution of radio- phosphorus in material obtained at au- topsy of leukemic patients indicated that selective localization took place in the bones and bone marrow. The content of labelled phosphorus in tissues infiltrated by leukemic cells was as high or higher than the uptake in uninvaded tissue which normally metabolizes phosphorus rapidly. A direct comparison of normal and leuke- Fig. 5. Distribution of radio-phosphorus in leu- kemic blood, showing the relatively greater uptake of mic tissues in human beings is, of course, the labelled phosphorus in the leukocytes as com- not possible; but according to data from pared to the erythrocytes and plasma. animal and clinical studies it appears probable that the pattern of selective reduction in the number of erythrocytes accumulation of radio-phosphorus in the was observed, although it was considerably infiltrated tissues of patients with leukemia less pronounced. The animal that had is similar to that observed in leukemic received 0.45 millicurie per pound was animals. sacrificed at the end of four weeks. No Scott and Cook (29), studying the effect gross abnormalities of the tissues were of large doses of radio-phosphorus upon seen. The monkey which had received , the cells of the circulating blood in chicks, 1.04 millicuries per pound died forty-seven observed a slight but definite decrease in days later. Marked hemorrhagic changes the polymorphonuclear leukocytes of the in the marrow as well as hrrhagic blood. Their results indicated the effect areas under the skin and in the intestinal of the irradiation from the selectively tract were noted. The remaining two accumulated radio-phosphorus upon the animals, which had received 0.71 and 0.76 bone marrow. Similar studies were con- millicuries per pound of body weight, ducted by Scott and Lawrence (30), in respectively, survived. At the end of order to obtain additional information approximately one year after the adminis- . regarding the effect of radio-phosphorus tration of the radio-phosphorus, they were upon the cellular elements of the blood apparently normal in every respect. Their when lethal and sublethal doses were blood pictures approached the normal employed. Four young monkeys (Mucacus pattern three months after the adminis- rhesus) were used in these experiments. tration of radio-phosphorus and at the The radio-phosphorus was administered end of a year they were essentially the intraperitoneally in the form of a sterile same as they had been at the beginning of isotonic solution of di-sodium phosphate. the experiments. Complete blood counts were taken before The logical development of the investi- the administration and at frequent in- gative work with radio-phosphorus de- tervals thereafter. The doses employed scribed in the preceding paragraphs was were 0.45, 0.71, 0.76, and 1.04 millicuries the therapeutic application of radio-phos- per pound of body weight. A marked phorus in the treatment of leukemia and depression of the number of leukocytes allied diseases such as polycythemia vera, in the blood was noted in all four animals. multiple myeloma, and metastatic cancer The maximum effect took place at from (7, 31, 32). This report does not under- two to four weeks after the radio-phos- take to evaluate the efficacy of the clinical phorus had been given. The relative therapeutic procedures but according to changes in the numbers of lymphocytes information thus far available radio-phos- and granulocytes were similar. Also a phorus is at least as effective as x-ray in

1 IOtr355 ... _-~ ...... -

550 JOSEPH G. HAMILTON November 1942

Fig. 6. Radio-autographs of the distribution of radio-phosphorus in the fruit and leaves of the tomato plant, the light areas being the regions where the great- est accumulatioti of radio-phosphorus took place. The fruit at various stages of development was retnovcd from a plant other thaii the one whose leaves are shown, riirie days after the atltnitiistration of radio-phos- phorus. The vertical coluniti at the left represents the radio-autographs from small greeii fruit, the lower three horizontal rows wcre taketi from sections of a large grerii fruit, atitl thc upper two rows represent scctioiis froin a fully ripe fruit. The iiitctisity of tlic ittiages is proportional to thecoticelitratioti of the radio-phos- phorus accuinulated it) thc different samples of fruit siiice this was all takeii frotii the same plaiit at thc saiiic tiiiie. The lenvcs wcre retnovetl from the plaiit thirty-six lioiirs after the iiitrodiictiotl of the lal)cIktl phohphxte into the ilutriclit Sdutioll. (Courtesy of Doctor Perry Stout, L'iiivcrsity of Califoriiia.)

the treatment of chronic. myclogeiious iii this rcspwt is dcfinitely superior to leukemia, chronic lymphatic leukemia, and x-ray thcrapy . polycythemia vera. 'l'hc. ~LtliiiiiiistratioII Stout aiid his co-workers (33) have of radio-phosphorus to 1cukc.mic patients followed tlic inetabolisni of inorganic phos- is not attended hy ratliatioii sickiicss and pliatcs iii the Icaves ant1 fruit of the Vul. 39 RADIOACTIVETRACERS IN Bro~.oc;u*\NII M~zn~cINrs t5.5 1 tomato plant by use ol radio-phosphorus and the technic of radio-autography. These workers administered the radio- phosphorus to roots of the plants as di-sodium phosphate and after a period of several days removed the leaves and fruit. The leaves and thin slices of the fruit were placed on photographic films and left together for a suflicient interval of time to allow the radio-phosphorus beta rays to leave images of the distribution of the accumulated labelled phosphorus in the specimens. The radio-autographs shown in Figure G indicate that the assimilated phosphorus was accumulated in the conduction system of the leaves and in the seeds of the green fruit. The ripe fruit can be seen to have accumulated small traces of the administered phosphate, but in this instance no detectable storage took place in the seeds, which suggests that in the ripe fruit the development of Fig. 7. Radio-autograph showing the capacity of the 1 the seeds had been completed before the intact leaf to absorb labelled di-sodium phosphate and radio-phosphorus was given. the circulation OF the absorbed phosphate into adjacent leaves. The dotted lines indicate the outlines of the These workers have employed radio- leaves. The white area at the left is the region where phosphorus to study the mechanism of the the solution containing the radio-phosphorus was painted on the leaf. The light streaks passing down the upward movement of inorganic ions in central axes of the leaves represent the path of circnla- cotton, willow, and geranium plants (34). tion of the absorbed phosphate. Following the administration of the labelled phosphate to the roots of the The results of these investigations indicate plants the bark was carefully detached that both the anions and cations of salts from the wood and held away from it with assimilated by plant roots are normally the aid of small strips of waxed paper. transported to the aerial portions of the When it had been demonstrated that a plants by way of the wood. Furthermore, considerable fraction of the radio-phos- such studies have demonstrated that the phorus had moved from the roots to the cells of the bark draw their mineral aerial parts of the plant above the region nutrients from the conducting vessels in where the bark had been detached, the the wood. plants were harvested and the stripped Stout and his associates (35) have con- section of bark and underlying wood were clusively demonstrated that assimilated examined for radioactivity. It was found phosphorus can move downward as well that there was very little radio-phosphorus as upward in the aerial portion of the in the detached portions of the bark, while tomato plant. This was shown by growing the wood directly beneath had as much as roots on the aerial portion of the stem two thousand times more. The bark midway between the top of the plant and nearby, which had not been separated from the normal root structure. These side the wood, had about the same content of roots were introduced into a nutrient the labelled phosphorus as did the wood. solution containing radio-phosphorus as Similar results were observed when radio- di-sodium phosphate and after several active isotopes of sodium, potassium, and hours the side roots were excised. The bromine were employed in these studies. various portions ol thc plant above and 552 JOSEPH G. HAMILTON November 1942

The metabolic processes of insects have been poorly understood because of the minute size of the organs, which has made it difficult to study their functions by direct biochemical methods. Radioactive tracers can be employed as an approach

d to this problem since the tracer technics are so much more sensitive than any of the classical chemical analytic procedures. In many instances the complete and quanti- tative collection of the excretory products and the dissection of the organs in the smaller insects are very difficult. For this reason radio-autography appears to be a very promising investigative method in this field. The tagged element or com- Fig. 8. Photomicrograph of a longitudinal section and its corresponding radio-autograph from a wax moth pound can be administered either larva, in the region of the mid intestine. The radio- or parenterally and subsequently the in- autograph shows the distribution of the labelled phos- phorus in the tissues of the insect, with selective Sects can be sacrificed and sections of the sition in the walls of the digestive tract, the silk glands, tissues prepared from which radio-auto- and the ducts of the silk glands, indicating that these organs were in a stage of rapid development, with a high graphs are made. Craig (37) was the demand for administered phosphorus. first investigator in the field to employ this technic and Figure 8 will serve as an the side roots were examined for example of the type of results which can radio-phosphorus and it was found that a be secured from this approach to the small but readily measurable portion of problems of insect metabolism. In this the assimilated phosphate had moved in the downward direction and had even particular experiment radio-phosphorus entered the normal root structure and the was administered two weeks before the t nutrient solution at the base of the plant. insects were sacrificed. The insects used This phenomenon of downward movement were the larvae of a small silk-producing of minerals in plants was also demon- moth and the longitudinal section shown strated by these workers in another experi- in Figure was taken from the merit (36). In this instance a few drops portion of the larva, which is about 3 of di-sodium phosphate solution containing mm. in diameter. The corresponding radio-phosphorus were painted on the sur- radio-autograph indicates that the Phos- face of a willow leaf. After several days phorus Was selectively accumulated in the &e leaf and those adjacent to it were walls of the digestive tract, silk glands, removed from the plant and the distri- and ducts of the silk glands. The selective bution of the labelled phosphorus demon- deposition of the labelled phosphorus in strated by the radio-autographic technic. these areas indicates that these organs were In Figure 7 the white area to the left is due developing rapidly and hence had a greater to the large content of radio-phosphorus demand for the administered Phosphate. at the point where the solution was painted The applications of tracer technic with on the leaf. The dotted lines indicate the the aid of artificial radioactivity to the outlines of the leaves and the light streaks problem of development of more effective passing through the central axes of the insecticides is of considerable importance, leaves represent the tagged phosphorus since it will be possible to tag various which was absorbed through the intact compounds employed in pest control and surface of the leaf and spread to all direc- observe directly the localization of these tions from that point. substances in insect tissues. I( )DINIS ccrt:iiri clcments, hut tioiic approaches the The thyroid gland is unique in that it capacity of the thyroid. It is not sur- has the ability to accumulate iodine prising, therefore that radio-iodine was selectively in relatively large quantities. accepted eagerly as a new research tool by This property is particularly striking be- thyroid physiologists. cause the iodine content of the blood Hertz and his associates (38, 39) first

Fig. 9. l'cchiiic eiiiploycd for the Iiiwsurcincnt of the uptake of ratlio- iotliiie by the thyroitl glaiitl 1'11 silrr. 'l'hc couritcr tiil~was placed agai!lst the thyroid glaiitl to tletcriniiie tlic gaiiiiiia ratlintioii froni thc ~CCIIIIIII- latetl radio-iotiiiie. averages less than one part in ten million tleinonstratcd the rapiditv with which while the thyroid nonnal1~-contain5 all- iodine is acci~rnulateclin the thyroid by the proximately one part in a thousatid ;lid of radio-iodine. In their initial experi- Therefore, the thyroid can conceritratc iiients they found significant accumulation the iodine it receives from the l~loodI)y ;I of iodine in the thyroids of rabbits within factor of ten thoiisand. Many organ\ arc a fcw rniiiutes after the atlrriinistration of capable of the wblc.ctivc* deposition of thc tag:getl clcriiciit. hIoreover, they

J 554 JOSEPH G. HAMILTON November 1942

noted that the uptake was much greater may be attributed in part to the apparent in the thyroids of animals with hyper- nced for additional iodine by goiters of thyroidism. this type. The form of the uptake curves These observations were confirmed in suggests that the mechanisms of iodine patients with hyperthyroidism by Hamil- accumulation and storage by the thyroids ton and Soley (40, 41). They compared were similar in these two groups. The the uptake of labelled iodine by the prompt accumulation of labelled iodine thyroids of normal controls and of patients by the thyroids of patients with hyper- with various types of thyroid disease, in thyroidism was followed by a rapid loss situ. This was accomplished by measuring of from one-half to four-fifths of the iodine, the gamma rays from the atoms of radio- which had been taken up during the first iodine that had been accumulated in the few hours of the experiments. These thyroid. A solution containing from 24 results were interpreted as indicating not to 100 microcuries of radio-iodine and a only that the hyperactive thyroid cells total of 14 mg. of iodine in the form of had a marked avidity for the accumulation sodium iodide was administered orally to of iodine, but also that the mechanism for each subject. None of the patients and the retention of the recently deposited normal controls had received iodine before iodine had been altered. The small uptake the labelled substance was given, and no of iodine by the thyroids of patients with additional iodine was administered until hypothyroidism may be explained by the the experiments were concluded. The fact that the thyroid tissue was unable to radioactivity of the thyroid of each subject supply a sufficient amount of the thyroid was determined by placing a Geiger hormone to meet metabolic requirements; counter tube against the neck and directly in other words, the failure of these thyroids over the isthmus of the gland, as is shown to accumulate iodine apparently was re- in Figure 9. Frequent determinations of lated to their inability to synthesize the the labelled iodine content of the thyroids thyroid hormone. were made over varying periods of time These experiments were repeated (42) ranging from five days to four weeks. The with a smaller group of patients and normal radioactivity of each thyroid in sih~was controls under the same conditions with compared to the radio-iodine content of the exception that each subject received a fraction of the sample given to the sub- a test dose which contained a total of jects. From these data the proportion of approximately 0.1 microgram of iodine. the administered dose taken up by the The uptake curves for this group are shown thyroid was calculated. in Figure 11. A marked increase in uptake Normal controls and patients with occurred in all four clinical types. This hyperthyroidism, non-toxic goiter, and result was expected since the total iodine hypothyroidism were studied by the aid content of the thyroid is relatively small of this technic. The results of these and its capacity to accumulate large experiments are summarized in Figure 10. amounts of iodine obviously is limited. The ordinate indicates the percentage of In patients with hyperthyroidism the up- uptake of the administered iodine by the take curves failed to repeat the form they thyroids, and the abscissa represents the took in the previous study. time in days after the labelled iodine had The increased uptake of radio-iodine in been given. The uptake curves illustrate all groups in which the smaller dose of the characteristic pattern of iodine accu- iodine was used emphasizes the importance mulation by the thyroids of the four of avoiding the dilution of the radio- clinical types included in this series. The element with the stable form of the element relatively large uptake of labelled iodine when it is desirable to prevent the physi- by the thyroids of patients with non-toxic ological action of flooding the tissues with goiter as compared to the normal controls the administered material. The results f

E e

550 JOSEPH G. HAMILTON November 1042

of these experiments revealed two effects received the 14-mg. test dose. The sample when the smaller test dose of iodine was of removed tissue, which was composed of employed: first, the uptake was remark- both normal and malignant thyroid tissue, ably elevated ; secondly, the metabolic weighed 204 gm. and had taken up 0.30 pattern was entirely different in the group per cent of the administered iodine. This of patients with hyperthyroidism. The is 0.001!3 per cent uptake per gram of larger test dose of iodine was employed tissue and is similar to the values obtained in the first series because it was deemed in the dissected cancers. In the patient necessary to compare the iodine metabo- who had received the 0.1-microgram test lism of the thyroid in different clinical dose, likewise the cancer could not be states under these experimental conditions, separated from the normal thyroid tissue. I since iodine is extensively used in the A comparison of the uptake of radio- treatment of both endemic goiter and iodine in normal and cancerous regions of 1 hyperthyroidism. the gland by radio-autography revealed

TABLEI : RADIO-IODINEUPTAKE OF THE THYROIDSAND OF TUMORTISSUE IN 2 PATIENTSWITH CARCINOMAOF THE THYROIDAFTER ORAL ADMINISTRATION OF 14.0 MG. IODINECONTAINING RADIO-IODINE Radio- Iodine Radio- Total Total Iodine per Iodine Weight Iodine Uptake Gram Uptake of in in of per Gram Diagnosis Tissue, Tissue, Tissue, Tissue, of Tissue. Gm. Mg. Per Cent Mg. Per Cent I Carcinoma of Thyroid A. Thyroid tissue 5 1.2 1.2 0.24 0.24 B. Cancerous tissue 138 <0.2 0.13 <0.002 0.001 C. Regional metastases 139 <0.2 0.05

A study of the localization of radio- that the malignant areas took up very iodine in cancerous thyroid tissue has been little of the labelled iodine. limited to four cases, in none of which a The technic of radio-autography has significant deposition of the labelled iodine lent itself readily to the investigation of in the malignant areas of the thyroid the relationship between the distribution occurred (42). Three of these patients of recently accumulated iodine and the received the 14-mg. test dose of labelled histologic structure of the thyroid tissue iodine and the fourth was given the 0.1- (43). The distribution of the accumulated microgram test dose. The thyroids were radio-iodine in thyroid tissue was studied removed two days after the administration in the following manner. A solution of of the tagged iodine. It was possible to from 100 to 1,000 microcuries of radio- dissect the cancerous portions from the iodine containing 14 mg. of iodine as invaded areas in two of the patients who sodium iodide was administered by mouth I7 had received the 14-mg. test dose. The to patients whose thyroid glands were total iodine content and the proportion of removed two days later. The thyroid the accumulated labelled iodine were deter- tissue was “fixed” in a 10 per cent solution mined in these samples. The results are of formaldehyde for twenty-four hours and summarized in Table I. These data indi- embedded in paraffin. Thin sections of the cate that the malignant portions of the impregnated thyroid tissue were cut with thyroid had no significant capacity either the aid of a microtome, and the cut to take up or to retain iodine. The cancer sections, which ranged from 3 to 5 microns could not be dissected from the uninvaded in thickness, were mounted on large glass thyroid tissue in the third patient who had slides. The mounted sections were washed i

Fig. 17. A photomicrograph of the thyroid and parathyroid tissue from a dog which received 300 microcuries of radio-iodine per kilogram 100 days before the tissue was removed. The parathyroid gland, shown in the lower right hand corner of the reproduction, was entirely normal histologically ; the surrounding thyroid tissue has been completely destroyed and has been replaced by fibrous tissue without any evidence of regeneration. f Vol. 39 RADIOACTIVETRACERS IN nI0LOGY AND MrsnIcrNIc 557

Fig. 12 (above). Photomicrograph of a section of iiormal thyroid tissue and its radio-autograph (X60). As in Figures 13, 14, and 15, the areas of darkening of the radio-autograph represent the regions of greatest accumulation of radio-iodine in the accompanying section of thyroid tissue. Fig. 13 (below). Photomicrograph from a section of hyperplastic thyroid tissue and its radio- autograph (X120). The large grayish areas in the section on the left represent colloid; the remain- ing acini are devoid of colloid. The radio-autograph indicates that most of the accumulated radio-iodine was stored in the colloid.

with xylene to dissolve out the paraffin of darkening on the radio-autographs and then were dipped in a dilute solution of represented the regions in the sections in collodion and set on edge to dry. By this which the greatest accumulation of the technic all the paraffin was removed and labelled iodine had taken place. Typical the sections were left protected by a film sections of normal thyroid tissue, non- of collodion approximately 1 micron in toxic goiter, thyroid hyperplasia, and thickness. The prepared sections were cancer of the thyroid are shown in Figures placed on Agfa no-screen x-ray film and 12, 1:3, 14, and 15. were held in close contact with the film Figure 12 was secured from a sample of by means of a small press. After a suit- norinnl thyroid tissue ; the radio-auto- able period of exposure2 the films were graph indicates that the accumulated removed and developed, and the sections radio-iodine was evenly distributed were stained with hematoxylin and eosin. throughout the section. The degree of The histological structure of each section resolution of the radio-autograph was not and its corresponding radio-autograph were sufficient to distinguish the cells indi- compared under a microscope. The areas vidually, but apparently the difference ___ lxtwcen the amount of tagged iodine in * stisfactory radio-autographs usually may be ob- tained from sections of plant and animal tissue which the cells and in the colloid was slight. contain a sufficient number of radioactive atorns so that The section shown in Figure 13 was ob- a total of 2 X 106 beta particles per square ccnlimeler will strike the film during the exposure. This value is tained from hyperplastic thyroid tissue. for radio-elements which emit hcta particles with an 'The section reveals a marked degree of average energy in excess of 150 Kev. The period of exposure, of course, depends upon the strength of the cellular activity and little colloid is present. sample, which in turn detemiines the interval of time Thc radio-autograph indicates that the required to obtain the necessary number of beta particles. small amount of remaining colloid had a I I iOb3b4 Fig. 14 (above). Photomicrograph of a section of thyroid tissue and its radio-autograph from a patient with a no~~-toxicgoiter (x60). Here the acini are enlarged and distended with colloid which had accumulated very little of the administered radio-iodine. The cells and small acini surrounding the large colloid deposits had a much greater ability to store the labelled iodine. Fig. 15 (below). Photomicrograph of a section and its radio-autograph from a patient with a cancer of the thyroid (XGO). The diffuse cellular area covering the right half of the section is made up of cancerous thyroid tissue. To the left are three small islands of uninvaded thyroid tissue which accumulated most of the radio-iodine. much higher proportion of the accumulated A definite correlation apparently exists radio-iodine than the cells of the adjacent between the relative uptake of radio- acini. This suggests that when a marked iodine in thyroid tissue and the degree of degree of thyroid hyperplasia takes place, hyperplasia shown by the histologic speci- part of the iodine accumulated in the mens. This relationship was striking in thyroid cells moves rapidly into the colloid all the sections obtained from patients while the remainder is returned by the with non-toxic goiter and with carcinoma, cells into the blood stream. Figure 14 in which the uninvaded tissue had under- shows section and radio-autograph from gone a compensatory hyperplasia. The a patient with a non-toxic goiter. The results of these studies suggest that hyper- accumulated radio-iodine in this instance plasia of thyroid cells is associated with an was stored predominantly in the cells and increased ability of these cells to concen- small acini which surround the larger trate the administered iodine from the acini. Figure 15 shows the difference blood stream. This phenomenon ap- between the deposition of iodine in rel- parently takes place when the thyroid atively normal thyroid tissue and in cells undergo hyperplasia and is independ- cancerous tissue. The obvious absence ent of the clinical condition which causes of appreciable radio-iodine in the neo- the hyperplasia. Here again the relation- plastic areas indicates that in this instance ship is demonstrated between the rate the thyroid cells which had undergone of turnover of an element in a tissue and malignant changes had lost their ahility its degree of selective deposition. to accumulate iodine. Chaikoff and his colleagues (34) made

-- . a detailed investigation of the metabolism end of twenty-four hours. A definite of labelled iodine in several types of experi- parallelism was noted in the loss of radio- mental animals, They compared the up- iodine by these tissues and by the blood, take of tagged iodine in the thyroids of which was interpreted to indicate that the rats with three different quantities of administered iodine moved in and out of 1 total iodine. Each animal in the first the tissues by simple diffusion rather than group received 0.5 mg. of labelled iodine. by a process of retention which apparently In the second group the individual dose takes place in the thyroid. was 0.03 mg., and in the last group ap- These same workers (45) also studied proximately 0.001 microgram was used. the rates of formation of thyroxine and P These values expressed in terms of total di-iodotyrosine by the thyroid. Accord- iodine per kilogram of body weight are ing to Harington (&)> these two com- approximately 2.5 mg., 0.15 mg., and 0.005 pounds can account for all the organically F microgram, respectively. In the first bound iodine found in the thyroid. The group the greatest uptake of radio-iodine metabolism of both thyroxine and di- in the thyroid was 2 per cent, in the second iodotyrosine has been investigated by group it was 7 per cent, and in the third numerous workers in the field of thyroid group it was 65 per cent. The results of physiology and has been reviewed by these experiments are similar to those ob- Salter (47) and Elmer (48). The applica- I tained in studies on human subjects. tion of radio-iodine to this problem has The thyroids of the normal human sub- made it possible to observe the rates of jects who received 14 mg. of labelled iodine synthesis of these two organic iodine com- (about 0.25 mg. per kilogram of body pounds from recently administered in- weight) took up approximately 4 per cent organic iodides. Rats and sheep were of the administered dose; the thyroids of used in Chaikoff’s experiments. The those who received 0.1 microgram of distribution of labelled iodine in the form i labelled iodine (about 0.002 microgram of di-iodotyrosine, thyroxine, and in- per kilogram of body weight) showed an organic iodine was determined following uptake of 20 per cent. The amounts of the administration of a solution of sodium labelled iodine in these instances were iodide which contained radio-iodine. A I similar to those administered to the second total of approximately 0.005 microgram of and third groups of rats and the accumu- labelled iodine was injected intraperitone- lation of labelled iodine in the thyroid ally into each of six sheep. Three of the ani- followed a similar pattern in both normal mals were sacrificed at the end of four hours human subjects and animals. and their thyroids were removed. The thy- Chaikoff and his co-workers also deter- roids of the remaining three animals were mined the distribution of radio-iodine in secured twenty-four hours after the admini- the tissues of the rats which had received stration of the labelled iodine. Inert the 0.001-microgram test dose. The maxi- potassium iodide, thyroxine, and di-iodo- mum concentration of labelled iodine in tyrosine were added to the samples of all the tissues (kidneys, testes, blood, skin, thyroid tissue to act as “carriers” to brain, liver, adrenals, and muscle) ap- facilitate the efficient separation of the peared five hours after the administration three radioactive iodine fractions from the of the radio-iodine. At the end of twenty- thyroids. Their chemical separation was four hours the levels of radio-iodine in the accomplished by a modification of the tissues, with the exception of the thyroid, procedures developed by Leland and Fos- fell to approximately one-tenth of these ter (39) and by Blau (50). The radio- values. Thereafter the content of radio- iodine content of the separated constitu- iodine in the tissues diminished more ents was determined and the relative slowly, and at the end of eight days it was proportion of labelled inorganic iodine, less than one-half the level observed at the thyroxin. and di-iodotvrosine was calcu- 560 JOSEPH G HAMILTON November 1942 lated. Similar studies were conducted in in other tissues of the body. This assump- a large group of rats, each of which re- tion is justifiable since the total quantity ceived approximately 0.001 microgram of of iodine administered to each animal was labelled iodine. The thyroids were re- so niinute that it could not have produced moved at intervals of two, four, forty- a n~easurableincrease in the normal iodine eight, and ninety-six hours. Because the content of any of the tissues. At the weights of the rat thyroids ranged from prcsent time similar studies are being 10 to 21 mg., desiccated thyroid was nindc on a large group of normal and added to the samples to act as a “carrier” thyrotoxic guinea-pigs. Preliminary re- for the extraction of the Iabelled iodine sults indicate that the proportion of compounds. labelled thyroxine in the thyroids of the The results of these experiments re- thyrotoxic animals is several times higher vealed that the administered iodine accu- than in the normal controls. Fraction- mulated by the thyroid tissue of both ation of the iodine compounds of the blood rats and sheep was rapidly converted into in these two groups of animals revealed a organically bound iodine. In the sheep, marked elevation of the thyroxine fraction at the end of four hours approximately in the thyrotoxic animals. These results 6.5 per cent of the labelled iodine in the are understandable, since the thyroids of thyroids was bound as thyroxine, 86 per the thyrotoxic animals were producing an cent as the di-iodotyrosine fraction, and excessive amount of thyroid hormone, the remaining 7.5 per cent as inorganic which apparently caused the elevated iodine. At the end of forty-eight hours the levels of labelled thyroxine. I relative proportion of thyroxine and in- Radio-iodine with the aid of radio- organic iodine was slightly higher, and the autography has been found a useful tool value of the labelled di-iodotyrosine was for the study of the embryologic develop- lower. The relative differences in distri- ment of the thyroid gland. Gorbman and bution of these three labelled iodine frac- Evans (51) undertook to determine at what tions were insignificant at the end of four stage of development of the tadpole of and forty-eight hours when compared the frog, Hyla regilla, the thyroid gland to the total uptake of labelled iodine by possessed the ability to accumulate iodine. the thyroids, which averaged slightly The tadpoles, which ranged in size from over 8 per cent at the four-hour interval the freshly hatched larva 7 mm. in length and 32 per cent at the end of forty-eight to the 38-mm. larva with well developed hours. The values obtained in the thyroids hind legs, were maintained for two days of rats differed somewhat from those in in 800 C.C. of water which contained 150 sheep. The labelled thyroxine fraction in microcuries of radio-iodine and approxi- the rat was from two to three times greater mately 1 microgram of iodine as sodium than that in the sheep, the di-iodotyrosine iodide. At the end of the two days the fraction was correspondingly lower, and tadpoles were fixed and serially sectioned. the inorganic iodine level was approxi- Radio-autographs were made from the mately the same in both animals. In sections, which were later stained with both sheep and rats the thyroxine fraction hematoxylin and eosin. A typical series was somewhat elevated during the latter is shown in Figure 16, representing the portion of the experiments and the di- various stages of the development of the iodotyrosine fraction was correspondingly tadpole. No evidence of iodine storage decreased. The inorganic iodine levels could be demonstrated in the larvae whose remained essentially the same through- total length was less than 10 mm. The out the experiments. These experiments first evidence of iodine deposition was demonstrate the rates of synthesis of di- noted in the 10-mm. larvae. At this stage iodotyrosine and thyroxine from the endo- the thyroid had two lobes and each lobe genous iodine available in the blood and consisted of from three to six follicles with _-

Vol. 39 RADIOACTIVETRACERS IN BIOLOGYAND MEDICINE 561

A B 10 mm. larva 11 mm. larva I.

C D 12 mm. larva 22 mm. larva Fig. 16. Radio-autographs of sections of the developing thyroids of tadpoles at various stages of development. The progressive increase in size and development of the thyroids is parallelled by a corresponding increase of their ability to accumulate the labelled iodine which is indicated by the radio-autographs.

$5 very little colloid. Each follicle was from vents which would have extracted any 10 to 15 microns in diameter and in cross- inorganic iodine in the tissues. d section was circumscribed by about four Later Gorbman and Evans (52) em- to six cells. As the tadpole increased in ployed labelled iodine to demonstrate that I size, the thyroid became larger, and the a protochordate possessed an organ which follicles, which were made up of more could accumulate iodine selectively. Or- cells and contained greater amounts of ganisms of this type do not have a thyroid colloid, increased in number. These de- gland but they possess a mucus-secreting velopmental changes were accompanied organ in the pharynx, the endostyle, which 4 by a parallel increase in size and intensity is considered to be the morphological of the radio-autographs, which indicated homologue of the thyroid. Small littoral a progressive ability of the thyroids to tunicates, Peraphora annocetens, were used accumulate more labelled iodine. The in these experiments. They were treated authors were of the opinion that the with radio-iodine, and radio-autographs labelled iodine in the thyroids of the tad- were made according to the usual technic. poles was probably bound in organic Contrary to expectations, the radio-au to- combination as thyroglobin before the graphs indicated that the endostyle con- radio-autographs were taken, for the sec- tained no detectable quantities of labelled tions had been treated with various sol- iodine. Large amounts of the substance, 562 JOSEPH G. HAMILTON November 1942 however, were accumulated in the septum The specimen was taken from a dog of the stolon (the reproductive organ that which had received the same dose of contributes to the formation of buds in radio-iodine per unit of weight and whose most protochordates which possess this thyroid was not removed until sixteen structure). The significant information weeks later. ‘The changes in the thyroids secured from these experimental studies of the other animals were similar and is that the generally accepted concept of also showed evidence of a marked degree the endostyle as the protochordate homo- of destruction. The quantity of radiation logue of the vertebrate thyroid probably is received by these thyroids is estimated erroneous. at between 15,000 and 25,000 roentgens. In another series of experiments Gorb- A careful examination of the other tissues man and Evans (53) studied the uptake of of these animals (spleen, liver, kidneys, labelled iodine in the thyroid glands of pituitary, parathyroids, adrenals, ovaries, fetal rats. The technic of radio-autog- and heart) revealed no pathological raphy was employed to determine the changes. Frequent blood counts were presence of accumulated radio-iodine in done on some of the animals that received the fetal thyroid tissue. The radio-iodine the radio-iodine, and no alteration of first appeared in the thyroids between the the blood picture was observed. At the eighteenth and nineteenth days of gesta- present time animals are being sacrificed tion-at the time when the fetal thyroid at varying intervals after the administra- tissue is undergoing the transition from a tion of radio-iodine in order to ascertain solid mass of closely packed cells to the more completely the character of the acinar type of structure which is the char- pathological changes. acteristic histologic pattern of adult thy- The results of the experiments with roid tissue. This observation was some- labelled iodine bring out three points of what surprising, since the total interval of interest in consideration of the possible use gestation in the rat is only twenty-one of radio-iodine in the treatment of hyper- days. It is interesting to note that the thyroidism. They show that the thyroids ability of fetal thyroid tissue to accumu- of patients with thyrotoxicosis accumulate late iodine and the initial appearance of and retain a large proportion of the ad- follicles take place at approximately the ministered radio-iodine; that the thyroid same stage of intra-uterine development. tissue can be destroyed selectively without A study on selective irradiation with apparent damage to the other tissues of radio-iodine in normal thyroid tissue of the body; that accumulated iodine is rabbits and dogs is now in progress (54). deposited selectively in the regions of the In these experiments, 300 microcuries of thyroid tissue which are the most hyper- radio-iodine per kilogram were admin- plastic. The areas of hyperplasia, to which istered. The total iodine content of each presumably the excessive elaboration of dose was kept below 0.1 microgram. (I131 the thyroid hormone is mainly referable, with a half-life of eight days was em- would be subjected to the greatest radia- ployed in these studies.) The purpose of tion from the beta rays of accumulated this work is twofold: first, to determine radio-iodine. The selective irradiation of the quantity of radio-iodine necessary to these areas should produce a relatively produce definite pathological changes in greater depression of the hyperplastic normal thyroid tissue; secondly, to deter- portion of the gland than of the normally mine if any other structures suffer damage functioning regions. It is deemed inad- from these relatively large quantities of visable to employ radio-iodine as a thera- radio-iodine. An example of the patho- peu tic agent in hyperthyroidism until logical damage to normal thyroid tissue the experimental studies on the action of produced by large doses of radio-iodine is large doses have been completed. Unlike shown in Figure 17. leukemia and other malignant disorders, IN AND Vol. 39 RADIOACTIVETRACERS BIOLOGY MEDICINE 5 6.7 hyperthyroidism is essentially curable, ological properties with those of i\>dine and the present methods of treatment are (5G). relatively safe. For this reason every A group of normal and thyrc+toxic precaution should be employed before a guinea-pigs received by injection a ~01~- potentially lethal agent, such as radio- tion containing both radio-iodine and iodine, is used to treat this relatively element 85. The excretion of thew two benign disease. The available experi- radio-halogens in both urine and feces mental information on the failure of was determined quantitatively. At vary- cancerous thyroid tissue to accumulate ing intervals the animals were SacHficed significant quantities of radio-iodine does and the content both of radio-iodin+ and not indicate that this agent will be of any element 85 in the tissues was meaQlred,

40

0 -THYROTOXIC. IODINE

0 - NORM-, IODINE I- - o, X -THYROTOXIC, ELEMENT 65 - =) 24 0 -NORMAL , ELEMENT 65 _------c---m - - X d /&------* -

11111111 11 111 10 20 30 40 50 60 70 TIME IN HOURS Fig. 18. Comparison of the uptake of radio-iodine and element 85 by the thyroid glands of normal and thyrotoxic guinea-pigs. value in the therapy of carcinoma of the The thyrotoxic animals were preparcd by thyroid. previous administration of thyrotropic hormone from the anterior portion of the ELEMENT 85 pituitary gland. Until its recent discovery by Corson, The uptake of these two radio-halogens MacKenzie and Segr6 (55), element 85, in the thyroids of normal and thyrotoxic the heavy homologue of iodine, was one of guinea-pigs (compared in Figure 18) was the missing members of the periodic sys- similar, a1 though the proportion of elrinent tem. The new element was prepared 85 accumulated by the thyroids ren)uined artificially by the transmutation of bis- consistently about one-third the value muth with the aid of the 60-inch Berkeley of the uptake of radio-iodine. The pro- cyclotron. Element 85 is radioactive and longed retention of element 85 in tlrvroid as far as is known does not occur in nature. tissue suggested that it was prOI,ably The chemical and physical periodicity of bound firmly in organic combinatioil and the elements is paralleled to a considerable that it may have been attached to hen- degree by their physiological properties. zene rings in a manner similar to iittline. For this reason it was felt that further The uptake of radio-iodine and elblllent proof of the identity of element 85 could 85 in other tissues was less than 1 per rent be secured by a comparison of its physi- of their concentration in the thyroid The 564 JOSEPH G. HAMILTON November 1942 rates of excretion of these two radio- the hemoglobin of the red blood cells. halogens were almost identical; the kid- Until these studies were made, the general neys acted as the chief channel of elim- assumption had been that the iron taken ination. into the body in food, water. and other sub- Since element 85 emits penetrating stances was absorbed in the stomach and x-rays, it was possible to study its accumu- small intestine and that the amounts of lation in the thyroid gland in situ by the this element absorbed in excess of the same technic as that employed to measure normal requirements were re-excreted in the accumulation of iodine. A solution the lower portion of the digestive tract. containing element 85 was administered In other words, the content of iron in the to a patient with a non-toxic goiter and body was believed to be regulated by the uptake was followed by measuring the excretion. radioactivity of the thyroid in situ. The In these studies with radio-iron, dogs

I

1 1 UPTAKE Or THE ELEMENT 85

BY THE THYROIO GLANO

IN A PATIENT WITH A NOM-TOXIC BOlfER.

12 4 6 8 l012l41~~8202224t6~ TIME IN HOURS. Fig. 19. Uptake of element 85 in the thyroid gland of a patient with a non-toxic goiter. The technic employed was the same as shown in Fig- ure 9. uptake curve resembled closely those were employed as experimental animals. obtained with labelled iodine in this type In the initial experiments Whipple and of thyroid disorder (Fig. 19). At the end his group (57, 58) compared the uptake of of twenty-four hours, approximately 10 radio-iron in normal dogs and in animals per cent of the administered sample was which had been rendered anemic by re- accumulated in the thyroid. Due to the peated bleeding and were maintained on a short half-life of element 85, it was not diet deficient in iron. Both groups of possible to continue this experiment for a animals received the labelled iron orally longer period of time. as inorganic ferric salts. They were sacrificed at varying intervals and the IRON radioactivity of the tissues was determined Whipple and his associates have em- with the aid of a Geiger counter. The up- ployed radio-iron as a tracer for the study take of the tagged iron was from 4.1 to of iron metabolism and, in particular, for 12.7 per cent in the anemic animals, while determination of the various factors relat- in the normal controls it ranged from 0.08 ing to the conversion of inorganic iron to to 0.24 per cent. In the anemic dogs most f

Vol. 39 RADIOACTIVETRACERS JN BIOLOGYAND MEDICINE 565

of the absorbed radio-iron was incorp- mately the same degree of radioactivity orated in the red cells of the blood, while in and containing the same amount of total the controls a much smaller proportion was iron as the two previous doses was ad- deposited in these cells. These studies ministered by mouth. At this time the indicated that the uptake of the admin- uptake was 10 per cent, while in the two istered radio-iron was determined by the preceding experiments it had been less need of the body for iron and that in the than 2 per cent. The authors suggest that anemic animals the total absorption of the the absorption of iron is not influenced labelled element and the relative propor- by the presence of anemia per se but is tion of the absorbed radio-iron in the controlled by degree of depletion of the blood were much greater than in the con- iron in the tissue and in particular by the trol animals. In later experiments (59) amount of iron present in the cells which these investigators studied the metabo- line the digestive tract. lism of tagged iron following its intravenous Austoni and Greenberg (63) investi- administration in normal and anemic dogs. gated the absorption, excretion, and dis- They observed that the rates of elimina- tribution of iron in rats which had been tion of the radio-iron were very slow in maintained on normal and iron-deficient both groups and that no significant differ- diets. The animals received the radio- ences in rate of excretion occurred. From iron in the form of ferric chloride by these observations they concluded that stomach tube in doses of from 2 to 8 mg. the body controls the level of its iron At varying intervals they were viviper- stores by regulating the quantity of iron fused to free the tissues from blood, and absorbed from the digestive tract rather the content of radio-iron in the blood, than by its capacity to eliminate the tissues, and excreta was determined. assimilated iron. In other studies Whipple These studies revealed that the normal and his co-workers (60, 61) showed that animals required about twelve hours for , a single dose of labelled iron to pass through i the absorbed radio-iron in the blood was i converted almost entirely into the iron- the stomach and small intestine. The containing protein, hemoglobin. They passage of radio-iron through the diges- 2 observed, moreover, that it was more tive tract was significantly slower in the efficiently converted into hemoglobin when animals maintained on iron-deficient diets. the total amount administered was very The uptake of the administered iron by small. the blood and tissues was greater in the In a recent experiment these investi- anemic animals. The specific accumula- gators (62) determined the iron uptake tion of absorbed iron per gram of tissue of a normal dog following the oral admin- was highest in bone marrow, blood, spleen, istration of radio-iron and found that 1.3 liver, and heart. The total accumulation per cent was assimilated. The animal was was greatest in muscle and blood; in the then rendered acutely anemic by the re- anemic animals, 25 per cent of the ad- moval of approximately two-thirds of its ministered radio-iron was stored in the total circulating blood. Twenty-four hours muscle and 14 per cent in the blood at the later the same amount of radio-iron was end of ten days. At the same time the given by mouth. The uptake of this dose corresponding tissues of normal animals of radio-iron was approximately the same contained only small traces of radio-iron. as that of the dose administered before This work confirms the observation of the animal had been bled. Sufficient time Whipple and his co-workers that the ab- was allowed to permit the hemoglobin level sorption of iron by the body is increased of the circulating blood to be replenished if the tissues have been depleted of their to a nearly normal value at the expense of stores of this element. It demonstrates the body stores of iron. Thereupon, a the significant fact that muscle tissue third sample of radio-iron of approxi- apparently acts as the chief storage depot HAMILTON November 1942

which is in the same group in the periodic system, is similar to that of calcium. Radio-strontium has a half-life of fifty- five days, can be readily prepared in relatively large amounts, and emits very energetic beta particles. Table TI indi-

TABLE11: DISTRIBUTIONIN THE TISSUESOF RADIO- ACTIVE CALCIUM,STRONTIUM, AND PHOSPHORUS TWENTY-FOURHOURS AFTER AN INTRAVENOUSINJEC- TION OF RADIO-CALCIUMLACTATE, RADIO-STRONTIUM LACTATEOR SODIUM RADIO-PHOSPHATE -____.___ Per Cent of Dose per Gram Wet Weight Skin Diges- and tive Other Bone Muscle Hair Tract Liver Viscera

I Ca46 22 0 33 0.20 0.36 0.12 0 23 Sr*9 12 0 17 0.15 0.23 0.07 0.13 1' Pa2 5.2 1.4 0.75 1.3 3 0 2 1 I I cates the distribution of phosphorus, cal- \ cium, and strontium in the tissues of mice Fig. 20. Difference in distribution of recently ab- when these substances were administered' sorbed radio-phosphorus and radio-strontium in rats i as shown by the technic of radio-autography. as inorganic salts. Although the propor- tion of strontium taken up in the body is lower than that of calcium, it will be for iron administered to animals with an noted that the selectivity of deposition of iron deficiency. these two elements in bone is almost the same. In Figure 20 a comparison between i CALCIUM AND STRONTIUM the distribution of strontium and phos- The bones of animals are composed phorus is made by the technic of radio- largely of calcium and phosphorus in the autography. In this experiment one of two form of tri-calcium phosphate. The con- animals received radio-phosphorus and the siderable interest in the metabolism of other radio-strontium. Two and a half calcium has stimulated investigators to days later the animals were sacrificed apply radio-calcium to metabolic prob- and sections of the entire bodies were lems. Campbell and Greenberg (G4), and prepared and placed upon films in order later Pecher (G),demonstrated by the to secure the radio-autographs. Figure use of labelled calcium that calcium is 20 shows that a moderate degree of phos- almost exclusively stored in the bones and phorus was accumulated in the skeleton that only small traces are distributed in and considerable quantities were deposited the soft tissues. This selective deposition throughout the soft tissues while almost of radio-calcium in the bony structures all of the strontium that had been assimi- suggested that it might be of therapeutic lated by the animal had accumulated in value in the treatment of malignant the bony structures. conditions which involve the bones. The In Figure 21 the distribution of radio- very long half-life of radio-calcium (180 strontium in soft tissues, normal bone, days), however, and the weak activities and tumor is shown in the radio-auto- that are available, definitely precluded graph. In this particular instance the its therapeutic application. Pecher ((is, soft tissue is comprised chiefly of muscle GO) predicted, and subsequently dernon- and skin, and the tumor is an osteogenic stratcd, that the Inetabolisni of strontium, sarcoma of the upper end of the tibia.

I lfib3'13 Vol. 30 RADIOACTIVETRACERS IN Bror,ocr AND MJUIICINR 567

’The patient received approximately 500 microcuries of radio-stron tium two days before the leg was amputated. A section approximately 5 mm. thick was made from the amputated extremity, and the radio-autograph secured from this tissue by placing it against the photographic film. The roentgenogram of the section was then prepared to visualize its struc- ture. As can be seen from the radio- autograph on the right, the strontium was chiefly localized in the region of the tumor and the more rapidly developing portions of the bones. The selective localization of the radio-strontium in the epiphyseal regions was to be expected since the patient was a child, and hence these regions were metabolically more active than the rest of the bone. The marked accumulation of strontium in the tumor indicates that the tumor cells were depositing the labelled strontium more rapidly than the surround- Fig. 21. Roentgenograni arid corresponding stron- ing uninvolved bone. tium radio-autograph of a thick section of an amputated leg from a patient with osteogenic sarcoma. The tumor. These studies indicate that radio-stron- which is indicated by the arrow on the roentgenogram tium, which is selectively deposited in has extended out into the surrounding soft tissue. The radio-autograph demonstrates that the radio-strontium bone and which possesses desirable radio- was selectively deposited in the tumor with small active properties (half-life, beta ray energy, amounts in the surrounding bone and that relatively and availability), may prove useful in little was accumulated in the soft tissues. clinical therapy. At the present time this phase of the problem is under investigation. attached to a large complex molecule. This reaction in the dark was found to be CARBON AND NITROGEN reversible in character and can be rcp- One of the outstanding examples of the resented by the following equation: RH application of a radioactive isotope as an + COX = RCOOH. It was suggested by indicator for fundamental research in the the investigators that this reaction rep- metabolic processes of biological systems resented the first step in the long and has been the use of radio-carbon by Ruben, complicated series of chemical reactions Kamen, and their co-workers to study which finally result in the formation of the metabolism of carbon dioxide in a sugars, starches, etc., by green plants. wide variety of organisms. These investi- The chemical identity of the molecule gators (67) demonstrated that plants and “R,” to which the labelled carbon diox- algae containing chlorophyll had the abil- ide is bound as a carboxylic acid, is not ity to assimilate a small but measurable known. The authors (GS) observed, how- Proportion of tagged carbon dioxide from ever, with the aid of the ultracentrifuge the air in the complete absence of light and diffusion measurements that the An extensive study of this phenomenon RCOOH molecule has a molecular weight of “dark reduction” in the green alga, of approximately 1,000 when it is extracted Chlorella pyrenoidostr., was carried out. from the alga hy the use of boiling water. The results of these studies indicated that In the ahsence of light all of the labelled assimilated carbon dioxide was re- carbon appeared to be in the carboxyl duced to a carboxylic acid which was group, which suggests that under these

I10b3-14 568 JOSEPH G. HAMILTON

conditions no further reduction of the are not synthesized directly in the plant assimilated carbon dioxide takes place. but that they are split off from large The “dark uptake” of the tagged carbon polyhydric molecules which are built up dioxide was very rapid when it was first on the enzyme “R.” In each step, how- introduced into the vessel containing the ever, the initial reaction involving each plants; but the rate of uptake diminished assimilated carbon dioxide molecule is its while the plants were maintained in the reduction to a carboxylic acid group, which atmosphere containing radioactive carbon is a non-photochemical reaction. The dioxide, and at the end of an hour very further reduction of the carboxylic radical little additional carbon dioxide was accu- involves photochemical reactions and re- mulated. This was interpreted to indicate quires the participation of both chloro- that the “dark uptake” of carbon dioxide phyll and light. was a reversible chemical reaction and at The mechanism for photosynthesis which the end of an hour equilibrium had been these authors have developed from their established with as many tagged molecules work with radio-carbon is at considerable leaving the plant as new molecules of the variance with the classical theory of gas from the surrounding atmosphere photosynthesis which has been accepted entered the leaves. by most investigators in this field. The When the experiment was conducted in latter concept postulated the following the presence of light the uptake of carbon type of reaction in the leaves of the plant: dioxide continued very rapidly and at the H20 + COP+ hv -+ HCHO + Oz,and the same rate from the time when the material subsequent polymerization of the formal- I was administered until the end of the dehyde to form more complex carbon com- experiment several hours later. Thus it pounds. A careful search was made for the was apparent that the assimilation of the presence of carbon compounds of low labelled carbon dioxide in the light was not molecular weight, including formaldehyde, a reversible reaction. Moreover, in the in Chlorella exposed to the tagged carbon presence of light only a small fraction of dioxide, and none was found within the the accumulated labelled carbon was found range of sensitivity of the method, which in the carboxylic radical ; apparently would have detected less than one part in most of the radio-carbon was converted a million of these substances had they to a more highly reduced state. In some been present. plants, as for example barley, approxi- Ruben and Kamen observed with the mately 20 per cent of the assimilated carbon aid of radioactive carbon dioxide that dioxide was synthesized into sugar within non-photosynthetic organisms (cells which two hours when the experiment was con- do not contain chlorophyll and do not ducted in the presence of light. utilize light in their metabolic processes) From these observations and the results had the capacity to reduce carbon dioxide of other experiments the authors suggest (69). They employed many different types the following type of reaction for the of microorganisms, including yeast cells, “light reduction” of carbon in the presence B. coli, and rat liver cells, in their experi- of chlorophyll, RCOOH + H20 + hv -+ ments and found that when these organ- RCHzOH + 0,. The RCHsOH molecule isms carried out their metabolic activities may take up carbon dioxide as did the in media to which labelled carbon dioxide original RH molecule, thus providing the had been added a significant portion of mechanism for a cyclical process leading the carbon dioxide was reduced and con- to the eventual synthesis of typical photo- verted into organic compounds such as synthetic carbon compounds by the step- acetic acid, propionic acid, etc. In some by-step addition of assimilated carbon cases a portion of the labelled carbon dioxide onto the enzyme molecule “R.” dioxide was found to have been incorpo- This mechanism implies that simple sugars rated into the cellular material, which is

I to Vol. 39 RADIOACTIVETRACERS IN BIOLOGYAND MEDICINE 569 made up of large and complex molecules. the discovery of artificial radioactivity to These observations are most striking since the medical sciences. The artificial radio- these organisms secure their energy by elements possess a dual potentiality : one, the oxidation of carbohydrates, fats, pro- in the field of pure research and the other teins, etc., and as a result carbon dioxide in that of practical therapeutics. In the is one of their chief metabolic end-prod- first place, these new substances may be ucts. Obviously, since such organisms employed as research tools to attack excrete carbon dioxide, the classical chemi- successfully the problems of metabolism cal and physical methods could never have that are closed to the classical procedures demonstrated that a small bul finite of chemistry and physics. These pre- proportion of the carbon dioxide n iolecules existing methods of metabolic research in their tissues are reduced. Furthermore, yield an essentially static picture of the Ruben and Kamen have shown that in complex biochemical processes which are some instances for every twenty-five to continuously taking place in every living fifty carbon dioxide molecules produced structure. The radioactive tracers, how- by oxidation one molecule was reduced. ever, give the investigator the opportunity The evidence thus far obtained has led to study the dynamic pattern of the chemi- to the suggestion that all living organisms cal integration which takes place in every have the capacity to reduce carbon di- living cell as it constantly is replenishing oxide. its substance from the surrounding en- Many organic compounds, such as vironment and discharging its waste prod- . sugars, acetic acid, propionic acid, lactic ucts. The biological action of the radia- acid, etc., are quite difficult to tag directly tions from the artificial radio-elements with radio-carbon by synthesizing them gives these substances their second im- in the test tube. These substances can portant rBle in medicine. This property be readily prepared, however, by ad- has given the clinician a new therapeutic ministering radioactive carbon dioxide to weapon for the treatment of neoplastic suitable organisms and after a period of an diseases. Radio-phosphorus serves as an hour or so the labelled compounds may be illustration of the application of a radio- separated from the biological material (70). element as tracer to the study of metabolic Mention should be made in this section problems and of its direct use as a thera- of the experiments by Ruben, Kamen, peutic agent of considerable promise in et a,?. (71) in which it was found that if the treatment of leukemia and allied barley plants are exposed to radio-nitrogen malignant states. a small quantity of labelled nitrogen Obviously, tracer research with the compounds is formed in the tissues of the artificial radio-elements is a necessary plants. The authors point out that this prelude to their therapeutic application. may be due to an exchange of the labelled Thus far only radio-phosphorus and radio- nitrogen with the nitrogen compounds in strontium have been subjected to clinical the plants. On the other hand, their trial and sufficient time has not elapsed observations may indicate that barley to permit an evaluation of the present plants can utilize small amounts of atmos- methods of treatment with these sub- pheric nitrogen for nutrition. The results stances. The potential usefulness of radio- of these experiments are of interest, since iodine in the treatment of hyperthyroidism there is considerable controversy con- is apparent and the time for its clinical cerning the fixation of nitrogen by non- test is almost at hand. In other words, this leguminous plants. report serves to indicate the various stages of development from the initial use of a CONCLUSION radio-element as a tracer to its therapeutic The material presented in this report applications. merely gives an idea of the significance of Attempts to secure the selective locali- 570 JOSEPH G. HAMILTON November 1942 zation of the artificial radio-elements have the primary aim. Since each of the 92 been limited in three directions. First, known elements has one or more radio- the radio-elements have been administered active isotopes and many of these have in the form of their simple inorganic com- half-lives that place them within the pounds. Secondly, the selective deposition range of therapeutic applicability, it is has been secured by what might be termed evident that the potentialities in the field normal physiological responses to the of selective irradiation far outweigh the metabolic demands of the tissues; these little that has been accomplished thus far. being the content of the element in the tissues, its rate of turnover, and the laying NOTE' Support for the work appearing under down of new tissues. Thirdly, the radio- the authot s name has been given by the American Cyanamid Company, the International Cancer active isotopes of only 21 elements have Foundation, and the Christine Breon Fund for been employed in biological research. Of Medical Research. this number only a few have been studied Crocker Radiation Laboratory intensively with selective localization as Berkeley, Calif.

APPENDIX

The following table, with the exception of the microampere hour of the bombarding ions. The yield data, was obtained from the tables given by term "microcurie" refers to the absolute number of Livingood and Seaborg in Review of Modern Physics disintegrations per second of the artificially pre- (72) and later by Seaborg in Chemical Reviews (73). pared radio-element; namely, 3.7 X lo4 disinte- All of the yields are given as microcuries per grations per second. All yields given which have a

Yield in Microcuries Radio- Type of Energy of Radiation in Mev per Micro- Produced Element Radiation Half-Life Particles y-Rays ampere Hour BY 30.0~. 0.013 No Y 0.1 Be-d-lHa. -" 21.0 min. 0.95 ..... 500.0* B-d-n >lo00 yr. 0.150 ..... 0.00005** N-n-p (be-d-nl 9.93 rnin. 1.2, 0.93 0.28 1000.o* C-d-n 3.0 yr. 0.58 1.3 1.0 Mg-d-a 14.8 hr. 1.4 1.46,2.0,3.03 10.000 Na-d-p 10.2 rnin. 1.8 0.9 500.0* Mg-d-p 14.30 days 1.69 No r 200.0 P-d-p 88.0 days 0.107 No 'Y 0.1 S-d-p 37.0 min. 1.1, 5.0 1.65, 2.15 1000.O* C1-d-p 12.4 hr. 3.5 ..... 50.0 K-d -p 180.0 days 0.2, 0.9 0.7 0.01 Ca-d-P 310.0 days ...... 0.85 1.0 Fe-d-oc 47.0 days 0.4, 0.9 1.0 0.03 Fe-d-p 270.0 days 0.4 (B+) 1.0 Fe-d-2n 0.58 (B-) 3000.0 Cu-d-p 12.8hr. { 0.66 (B+) '250.0 days 0.4 (B+) 0.45,0.65,1.0 0.5 Cu-d-2n 1.1, 1.7, 2.7, 1.5, 2.2, 3.2 10.0** As-n-y (Be-d-n) 26.8 hr. B-, 0.7, 2.6. Bf 1.3 (B-) ..... 2.0 Ge-d-n 16.0 days { 1.9 (B+) 34.0hr. 0.7 0.65 10.0** Br-n--y(Be-d-p) 18.0 days ...... 10.0 Rb-d-p 55.0days 1.50 No Y 7.0 Sr-d-p 8.0 days 0.687 0.4 20.0 Te-d-n 7.5hr. 6.0 ..... 1.0 Be-a-2n (32 Mev a s) ~ ~- adjoining column. The double-asterisk refers to the production of the radio-elements by neutrons re- sulting from the bombardment of beryllium by 16,000,000 electron volt deuterons. These yields from neutrons, which are expressed as microampere hours of deuterons, were estimated on the basis of the following quantities of material irradiated by the neutrons: 6C14 from 25 kg. of ammonium nitrate, &s76 from 500 gm. of cacodylic acid and 35BrS2 from 2000 C.C. of brom-benzene. The values given for the yields are in many in- stances approximations and are subject to large er- rors. The data for the yields of 11NaZ4,15P32, &e59, 39Srs9,and are estimated to be accurate to approximately 50 per cent. The remaining yield data are much less exact and are subject to con- siderably larger fluctuations in accuracy.

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