PHOSPHORUS METABOLISM OF THE SOFT TISSUES OF THE NORMAL MOUSE AS INDICATED BY RADIOACTIVE PHOSPHORUS '

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1 PHOSPHORUS METABOLISM OF THE SOFT TISSUES OF THE NORMAL MOUSE AS INDICATED BY RADIOACTIVE PHOSPHORUS ' H. B. JONES, I. L. CHAIKOFF, AND JOHN H. LAWRENCE (From the ~ivision of ~kysiology'of the Medical School and the Radiation Laboratory, University of California) In the course of investigations dealing with the phosphorus metabolism of neoplastic tissues, which are described in the following paper, it was necessary to compare the rates of deposition of radioactive phosphorus in such tissues with those in normal tissues obtained from the animal in which the tumors were permitted to grow. Although observations on the distribution of injected labeled phosphorus in the tissues of the rat (I) and mouse (2, 3) have appeared, a systematic study of the soft tissues of the mouse was required for a comparison under conditions identical for normal and tumor tissues. In the present investigation radioactive phosphorus was injected into mice and the distribution of the labeled phosphorus determined in such tissues as liver, kidney, small intestine, brain, blood, etc., at various intervals up to seventythree hours after the administration of P". Mice of the A strain, weighing between 21 and 25 gm., were used. The animals were permitted access to food and water at all times during the experiment. The diet consisted of a balanced mixture in pellet form supplemented with whole oats. Each mouse received intraperitoneally 0.2 C.C. of an isotonic solution of radioactive sodium phosphate (Na2HP0,) containing 3 mg. of phosphorus per C.C. A total of 5 microcuries ' was contained in the injected phosphate. The animals were sacrificed at various intervals thereafter and the following tissues were removed for analyses: liver, kidney, small intestine, blood, lymph nodes, lung, skeletal and cardiac muscle, and brain. Treatment of Tissue: Liver, kidneys, small intestine, and lungs were removed in toto and after being freed of adhering blood were reduced to a uniform paste in an agate mortar. Samples of the macerated tissues weighing between 20 and 100 mg. were placed in specially prepared cellophane cones held inside weighing vials. Skeletal muscle was removed from a single limb and buttocks, freed of visible fat and large nerves, reduced to a paste, and sampled as above. The heart was blotted free of blood, pounded into a paste, and the whole mass was put inside a cellophane cone. The brain was separated into right and left halves and each was transferred directly to a cone. Blood was obtained from the spurting arteries of the severed neck in such a 'Aided by grants from the Dazian Foundation for Medical Research. The assistance furnished by the Works Progress Administration (official project No , Unit A6) is gratefully acknowledged. 2 Determined by a uranium standard. 235

2 236 H. B. JONES, I. L. CHAIKOFF, AND JOHN H. LAWRENCE manner as to avoid contamination with tissue juices, and a small sample was transferred to the cone within the weighing vial. Lymph nodes were removed from the submaxillary, cervical, and axillary regions. They were shelled from their capsules and all of them transferred to the small cellophane cone. Measurement of Radioactivity: The cellophane cone, which measured not more than 2 cm. in length, and the tissue within it were placed upon a blotter measuring 3.5 X 6 cm. The blotter and its contents were completely sealed with a very thin cellophane wrapper. A glass vial was then used as roller and the contents of the cone were squeezed unifo;mly into a thin film over the entire 20 sq. cm. surface of the blotter. The blotter was allowed to dry over a period of several days and its radioactivity was then determined by means of a thin-walled aluminum Geiger counter, which has been described elsewhere (4). In no case did the amount of residue adhering to the blotter after drying amount to more than 1-3 mg. per sq. cm. of surface area and hence no correction for self-absorption of the B-emanations was deemed necessary. RESULTS Distribution per Gram of Tissue The distribution of labeled phosphorus in the soft tissues of normal mice is shown in Figs. 1, 2, and 3. The soft tissues of the mouse may be grouped into three classes, according to the level of phosphorus turnover as measured with radioactive phosphorus. Liver, kidney, and small intestine constituted the group with the highest activity per gram of,tissue. Next in order were cardiac and skeletal muscle, lymph nodes, and lungs. The group with the lowest activity included blood and brain. The results recorded here were obtained from 360 separate analyses made from the tissues of 20 mice. Each point in Figs. 1, 2, and 3 represents the mean of 4 to 6 closely agreeing values obtained from 2 or 3 mice. Liver: The highest concentration of radioactive phosphorus was found in the liver (Fig. I). Eight hours after the intraperitoneal injection of radioactive phosphorus, about 9 per cent of it was deposited in each gram of hepatic tissue. A rapid turnover of phosphorus takes place, however, for sixteen hours later the amount of labeled phosphorus still present is reduced to approximately half, namely 5.0 per cent, while seventy-three hours after the injection the labeled phosphorus content of the liver is only 2.7 per cent per gram of tissue. Phospholipids account for a considerable proportion of the total phosphorus deposited in the liver. About one third of the administered radioactive phosphorus is deposited in the liver as newly synthesized phospholipid (4). The rates of turnover of both labeled phosphorus and labeled phospholipid are roughly similar. The content of phosphorus and phospholipid rises and falls rapidly in the liver, the maximum of each being deposited between five and ten hours after the injection of the phosphorus. SmaU Intestine: The curve depicting the rate of turnover of labeled phosphorus in the small intestine resembles that for the liver (Fig. 1). Thus the maximum deposition occurred between two and eight hours after injection, but at this interval approximately 6.7 per cent of the phosphorus was found in each

3 PHOSPHORUS METABOLISM OF SOFT TISSUES 237 gram of small intestine. - At the rater intervals, namely fifty and seventy-three hours, there was practically no difference in the concentration of labeled phosphorus in liver and small intestine. Kidney: The curve for kidney parallels that of the small intestine fairly closely (Fig. 1). Thus the highest activity in the kidney was at two and a HOURS AFTER P' ADM IN1STRATK)N Fro. 1. LABELED PHOSPHORUS CONTENT PER GRAM OF LIVER, SMALL INTESTINE AND KIDNEY AT INTERVALS UP TO SEVENTY-THREE HOURS APTER INTRAPERITONEAL INJECTION OF RADIOACTIVE PEOSPEORUS The ordinate represents the percentage of the labeled phosphorus found per gram of tissue. half hours after the injection of the radioactive phosphorus, at which time 6.6 per cent was found in each gram of kidney. Skeletal and Cardiac Muscle: The phosphorus activities of skeletal and cardiac muscle are much lower than those of the three tissues recorded above. The maximum values found were below 5 per cent (Fig. 2) as compared with values between 6 and 9 per cent for small intestine, kidney, and liver.

4 238 H. B. JONES, I. L. CHAIKOFF, AND JOHN H. LAWRENCE The general shape of the curve for each type of muscle, however, resembles those of the more active tissues, with the maximum deposition occurring between five and ten hours after the injection of the radioactive phosphorus and a reduction in the phosphorus concentration to about haif the maximum at the fifty-hour interval. Lymph Nodes and Lung: The phosphorus turnover of lymph nodes is of the same order as that of skeletal muscle (Fig. 2). At the early intervals 0 MUSCLE LYMPH NODES 8 LUNGS ao I HOURS AFTER pz ADMINISTRATION FIG. 2. LABELED PHOSPHORUB CONTENT PER GUM OF HEART, MUSCLE, LYMPH NODES AND LUNGS AT INTEUVALB UP TO SEVENTY-THREE HOURS AFTER INTRAPERITONEAL INJECTION or RADIOA~ PHOSPHORUS The ordinate represents the percentage of the labeled phosphorus found per gram of tissue. after administration the rate at which radioactive phosphorus is incorporated into the lung tissue is definitely slower than in either muscle or lymph nodes. The maximum amount found in the lung was 2.6 per cent per gram of tissue. Each gram of lung showed more than twice the activity of blood, a finding of interest in view of the high content of blood in the lungs. Blood: Blood showed a surprising1y low activity (Fig. 3) when compared with such tissues as liver and kidney. Indeed, with the exception of the brain,

5 PHOSPHORUS METABOLISM OF SOFT TISSUES 239 its turnover of labeled phosphorus was lower than that of the other tissues examined. Thus at the ten-hour interval, a little over 1 per cent of the administered radioactive phosphorus was deposited per gram of blood. Brain: Not only did brain show the lowest activity of all soft tissues examined (Fig. 3), but the type of turnover differed distinctly from that of all the other tissues. In the latter, the maximum deposition of labeled phosphorus occurred before ten hours, after which a decline in concentration of variable HOURS AFTER P* ADMl NlSTRATlON FIG. 3. LABELED PHOSPHORUS CONTENT PER GRAM OF BLOOD AND BRAIN AT INTERVALS SEVENTY-THREE HOURS AFTER INTRAPERITONEAL INJECTION The ordinate represents the percentage of labeled phosphorus found per gram of tissue. UP TO OF RADIOA~IVE PHOSPHORUS degree occurred; at the fifty-hour interval the content of labeled phosphorus per gram of tissue was always below that observed at the five- to ten-hour interval. In the brain, on the other hand, there was a gradual accumulation of labeled phosphorus, and the maximum content was not observed until the twenty-four-hour interval. No decline occurred during the rest of the experiment, and even at the end of the period of observation, namely seventythree hours, the concentration of labeled phosphorus per gram of tissue was still as high as at the twenty-four-hour interval.

6 240 H. B. JONES, I. L. CHAIKOFF, AND JOHN H. LAWRENCE The percentages of the labeled phosphorus deposited in the whole organs of the mouse were compared at two intervals after administration of radioactive phosphorus, i.e. eight and fifty hours (Fig. 4). As noted above, maximum or nearly maximum deposition of phosphorus occurred at the earlier interval in all tissues except the brain. For the purpose of the comparison shown in Fig. 4, the content of labeled phosphorus in 1 grn. of muscle was taken.' The value for blood phosphorus was calculated on the assumption that blood is accountable for 7 per cent of the total body weight. Lymph nodes were obtained from three regions, cervical, submaxillary, and axillary. The values recorded in Fig. 4 probably represent half of the labeled phosphorus contained in such tissue of the normal mouse. The soft tissues with the exception of skeletal muscles retain about 22 per cent of the administered labeled phosphorus at the eight-hour interval, but so rapid is the turnover of phosphorus in most of these tissues that forty-two hours later the amount retained has been reduced to approximately 8 per cent. The organ that shows the largest amount of labeled phosphorus at both intervals is the liver, but whereas at the earlier interval the whole liver contains almost twice the amount present in the small intestine, there is but little difference in the amounts retained by these two organs at the fifty-hour interval. At the eight-hour interval over 40 per cent of the total amount of labeled phosphorus deposited in the soft tissues examined in this study is found in a single organ, the liver, which accounts for but 5.5 per cent of the total body weight. Although the kidney was listed above among the more active tissues, yet its rble in the phosphorus metabolism of the whole animal is minimized by the limited amount of this tissue present in the body. The bulk of the phosphorus deposited in the soft tissues is found in three organs: liver, small intestine, and muscle. At the eight-hour interval, these three organs contained approximately 90 per cent of all of the labeled phosphorus deposited in the various soft tissues examined. The deposition of labeled phosphorus in the liver, lymph nodes, and muscles of the mouse has previously been examined by Lawrence et al. (2, 3) at intervals between seventeen and one hundred hours after the administration of radioactive phosphorus; the results obtained compare favorably with those recorded here. These workers have also shown that bone continues to acquire labeled phosphorus for as long as twenty days. As regards another species, the rat, it is of interest that when the comparison is made in respect to whole organs, the relative distribution of phosphorus in the soft tissues, as determined by Cohn and Greenberg, is strikingly similar to that obtained here in the mouse. 8 The average weights of the whole liver, small intestine, both kidneys, heart, both lungs, and brain observed in a group of A-strain mice weighing between 22 and 24 gm. were respectively 1.22, 0.90, 0.32, 0.077, 0.11, 0.22 gm. The lymph nodes pooled from the submaxillary, cervical, and axillary regions weighed on the average gm. 4 Total muscle of the leg was used. If it be assumed that the labeled phosphorus is uniformly distributed throughout skeletal muscle and that this tissue represents approximately 40 per cent of the total body weight, then the total musculature contains approximately 33 per cent of the administered labeled phosphorus.

7 PHOSPHORUS METABOLISM OF SOFT TISSUES The phosphorus metabolism of the soft tissues of the mouse was investigated with radioactive phosphorus as indicator. Following the injection of PSa the distribution of the labeled isotope was determined at various intervals up to seventy-three hours. I LIVER 2 SMALL INTEST 'INE 3 MUSCLE 4 KIDNEYS 5 BLOOD 6 HEART 7 LUNGS 8 BRAIN 9 LYMPH NODES 8 HOURS AFTER P* 50 HOURS ADMINISTRATION FIG. 4. THE DISTRIBUTION OF LABELEO PHOSPIIORUS IN WHOLE ORGANS OF THE NORMAL MOUSE The ordinate represents the per cent of administered phosphorus found per whole organ. For the purpose of the present comparison, the content of labeled phosphorus in 1 gm. of muscle is shown. 2. With the exception of the brain, all tissues showed a characteristic response to the intraperitoneal administration of radioactive phosphorus. There was a sharp rise in the concentration of labeled phosphorus during the early hours; the' maximum deposition was observed between five and ten hours. In most tissues the loss of labeled phosphorus was less pronounced than the rise.

8 242 H. B. JONES, I. L. CHAIKOFF, AND JOHN H. LAWRENCE The brain showed a progressive increase in the amount of labeled phosphorus deposited, long after the other tissues had attained their maximum. 3. On the basis of the rate at which labeled phosphorus was deposited and removed per gram of tissue, the highest activity occurred in liver, small intestine, and kidney; next in order were cardiac and skeletal muscle, lymph nodes, and lungs; the lowest activity was found in blood and brain. 4. The bulk of the phosphorus deposited in the soft tissues soon after administration was found in three organs: liver, small intestine, and muscle. 1. COHN, W. E., AND GREENBERG, D. M.: J. Biol. Chem. 123: 185, LAWRENCE, J. H., TUTTLE, L. W., SCOTT, K. G., AND CONNOR, C. L.: J. Clin. Investigation 19: 267, LAWRENCE, J. H., AND SCOTT, K. G.: Proc. Soc. Exper. Biol. & Med. 40: 694, JONES, H. B., CHAIKOFF, I. L., AND LAWRENCE, J. H. : J. Biol. Chem. 128: 631, 1939.