STUDIES ON THE MECHANISM OF NITROGEN STORAGE

STUDIES ON THE MECHANISM OF NITROGEN STORAGE VI. RATE OF PROTEIN SYNTHESIS AND SIZE OF THE NITROGEN POOL* BY PAUL D. BARTLETT AND OLIVER H. GAEBLER WITH THE TECHNICAL ASSISTANCE OF BEVERLY CADY (From the Edsel B. Ford Institute for Medical Research, Henry Ford Hospital, Detroit, Michigan) (Received for publication, August 13, 1951) Studies on the rate of protein synthesis and the size of the nitrogen pool in both the rat and the human (1, 2) have provided the research worker with technical procedures and mathematical treatment by which it is possible to assess directly nutritional and endocrine effects on the regulation of amino acid and protein metabolism. Sprinson and Rittenberg (2) have shown that the empirical equation A& = ba(l - e--bt) describes the excretion of labeled glycine nitrogen in both the rat and the human and have developed the relationship of the constants A and B to the size of the metabolic nitrogen pool and the rate of protein synthesis. The recently published studies of Hoberman (3-5), in which rate-process constants for the protein e amino acid equilibrium and for the catabolism of amino acids to urine nitrogen are derived and tabulated, include a number of observations (3) which are of considerable interest in connection with the present experiments and preceding ones (6). Hypophysectomy of the rat was found to result in an increase in the rate-process constant for the catabolism of amino acids, and treatment of the hypophysectomized animal with growth hormone returned the constant to normal. Synthesis of protein from amino acids was shown to be only slightly affected by hypophysectomy, but definitely accelerated in the hypophysectomized animal treated with growth hormone. Doubt concerning the production of continuous growth with growth hormone has largely been dispelled since the demonstration by Evans et al. (7) that repeated injection of this hormone into normal adult female rats resulted in continuous growth for a period of 437 days. Parallel studies of the chemical composition of the body (8) showed that growth hormone produced a rise in protein and water content and a lowering of the fat content of the body. Since changes such as these are characteristic * This study was supported in part by a grant from the Michigan Chapter of the Arthritis and Rheumatism Foundation. 1

2 MECHANISM OF NITROGEN STORAGE. VI of normal growth (9), designation of nitrogen storage and gain in body weight induced by growth hormone as true growth was felt justified (8). It has been suggested that growth, which is characterized in one respect by the accumulation of body protein, may result from a depressed rate of protein or amino acid catabolism, by an accelerated rate of protein synthesis, or by a combination of these mechanisms. Quantitative evaluation of amino acid and protein metabolism in terms of the rate of protein synthesis, the rate of amino acid catabolism, and the size of the nitrogen pool in animals in nitrogen balance, and in animals in which gain in body weight and nitrogen storage have been induced through the administration of anterior pituitary growth hormone, should thus prove of interest in characterizing the over-all mechanisms involved in the production of nitrogen storage and the regulation of processes involved in growth. We have, therefore, undertaken to determine whether the empirical equation of Sprinson and Rittenberg (2) describing the excretion of labeled glycine nitrogen by the rat and the human can be applied to the dog, and, if SO, to determine the rate of protein synthesis, the rate of amino acid catabolism, and the size of the nitrogen pool in both nitrogen balance and nitrogen storage experiments. EXPERIMENTAL Animal-Adult female dogs weighing from 11 to 17 kilos, maintained in metabolism cages on Stock Diet 6 in the manner previously described (lo), were used in all studies. Procedures-In general, two types of experiments were conducted on each animal. In one experiment, the animal was maint ained in nitrogen balance on the required constant dietary intake of Stock Diet 6. Daily observations on the total urinary nitrogen excretion and body weight were taken as indicative of the stage of nitrogen equilibrium in the animal. In the other experiment, nitrogen storage was induced in such an animal by the subcutaneous administration of small daily doses of anterior pituit ary growth hormone. Growth hormone Preparation Rx099916l was used in two of the induced nitrogen storage experiments, and Preparation 22KRll was used in the third experiment. The lyophilized preparations were taken up in 0.8 per cent saline and injected in doses of 5 mg. per day for periods 1 Growth hormone Preparation Rx099916, with a reported potency of 3502 growth units per gm., was supplied through the courtesy of Dr. D. A. McGinty and Mr. L. W. Donaldson of Parke, Davis ana Company. Assays in 26 to 28 day-old hypophysectomized rats conducted in our laboratories showed the preparation to have a growth-promoting potency approaching that reported for pure crystalline growth hormone (10). Growth hormone Preparation 22KRl was supplied by Armour and Company and was reported to be approximately equivalent to their growth hormone standard Preparation 22KR2.

P. D. BARTLETT AND 0. H. GAEBLER 3 of 5 days. At the desired time during the experiment in which the animal was in nitrogen balance and on the 4th day of the induced nitrogen storage experiments, the diet was supplemented with 32.5 atom per cent excess N15-labeled glycine in doses of 10 mg. per kilo of body weight. Collections of urine were then made at 3, 6, 9, 12, 24, and 48 hours after ingestion of the labeled amino acid. Each collection was terminated by catheterization of the animal and washing of the bladder. Total urinary nitrogen excretion was determined in each collection by a semimicro-kjeldahl procedure. An aliquot of the urine was then analyzed for Nr5 by the procedure described by Sprinson and Rittenberg (11). Isotope ratios were 60-.* v) NITROGEN BALANCE z 50 - EXPERIMENT 0 aw - TIME IN HOURS FIG. 1. Rate of excretion of Nl5label during typical nitrogen balance and nitrogen storage experiments on Dog 55. determined on the Consolidated-Nier mass spectrometer, model 21-201.2 Calculations of the rate of protein synthesis and the size of the nitrogen pool were made according to the mathematical treatment of Sprinson and Rittenberg (2). Calculations of the rate of amino acid catabolism and the rate at which amino acids were transformed into protein were also made by the use of the mathematical treatment of Hoberman (3). Results The rate of urinary excretion of ingested N15 during typical nitrogen balance and nitrogen storage experiments on Dog 55 is shown in Fig. 1. 2 Nl5 analyses were performed through the cooperation of the Department of Chemical and Metallurgical Engineering of the University of Michigan. We wish to express our appreciation to Professor 12. R. White, Mr. Banchero, and Mr. David Brown for their splendid cooperation.

MECHANISM OF NITROGEN STORAGE. VI TIME IN HOURS -0.2-0.4-0.6 FIG. 2. Plot of data given in Tables I and II showing application of the empirical equation of Sprinson and Rittenberg (2) to the utilization of glycine nitrogen by the dog. Curves I and II are, respectively, for nitrogen balance and nitrogen storage experiments on Dog 44. Curves III and IV are, respectively, for similar experiments on Dog 55. Time hrs. m. o-3 1.13 3-6 1.62 6-9 1.69 9-12 1.28 12-15 0.73 15-24 1.88 2448 8.04 TABLE Rate of Utilization of Glycine Nitrogen by Dog Dog 44, nitrogen balance atom per cent excess 0.029 0.056 0.053 0.048 0.042 0.021 0.012 meg. I m.cq. 0.023 0.023 0.036 0.932 0.065 0.088 0.136 0.742 0.064 0.152 0.234 0.556 0.044 0.196 0.301 0.430 0.022 0.218 0.335 0.365 0.028 0.246 0.378 0.284 0.069 0.315 0.485 0.080 Dog 44, nitrogen storage O-3 0.45 0.023 0.007 0.007 0.011 0.956 3-6 0.76 0.035 0.019 0.026 0.040 0.844 6-9 1.08 0.036 0.028 0.054 0.083 0.679 9-12 0.75 0.032 0.017 0.071 0.109 0.577 12-24 1.67 0.024 0.029 0.100 0.154 0.407 2448 5.28 0.011 0.042 0.142 0.218 0.158 * xz = N16 excreted; XO = N16 fed at zero time = 0.65 m.eq. t A = 0.527 for N balance experiment (see Table IV); A = 0.258 for N storage experiment (see Table IV).

P. D. BARTLETT AND 0. H. GAEBLER 5 The excretion is very rapid during the first 24 hour period in the nitrogen balance experiment, approximately 35 per cent of the ingested N15 being excreted. During the second 24 hour period, the excretion rate falls very rapidly, amounting to only 7.0 per cent of the ingested N15. Production of nitrogen storage in this animal with growth hormone resulted in a marked reduction in both the rate and quantity of N15 label in the urine, only 19 per cent being excreted during the first 24 hours and 7 per cent during the TABLE Rate of Utilization of Glycine Nitrogen by Dog II Time hrs. Total N N S excretion concentration gm. o- 3 1.22 3-6 1.66 6-9 1.41 9-12 1.74 12-24 4.46 24-48 10.59 / Dog 55, nitrogen balance ltom fier cent excess 0.023 0.042 0.044 0.040 0.028 0.007 m.e*. 0.020 0.0.50 0.044 0.050 0.090 0.053 Dog 55, nitrogen storage o- 3 0.93 0.018 0.012 3-6 1.22 0.027 0.024 6-9 1.23 0.031 0.027 9-12 1.19 0.028 0.024 12-24 2.65 0.027 0.050 24-48 7.50 0.009 0.048?n.eq. 0.020 0.028 0.937 0.070 0.098 0.781 0.114 0.160 0.642 0.164 0.229 0.487 0.254 0.354 0.208 0.307 0.428 0.043 0.012 0.036 0.063 0.087 0.137 0.185 0.017 0.049 0.087 0.121 0.191 0.259 0.944 0.833 0.704 0.590 0.352 0.123 * XE = N16 excreted; X0 = N15 fed at zero time = 0.714 m.eq. t A = 0.447 for N balance experiment (see Table IV); A = 0.295 for N storage experiment (see Table IV). second 24 hour period. Total excretion of the N15 label during the 48 hour test period was 42 per cent in the nitrogen balance experiment and 26.0 per cent in the nitrogen storage experiment. Demonstration that the excretion of the labeled glycine nitrogen in the dog follows the empirical equation of Sprinson and Rittenberg (2) is shown in Fig. 2 in which log (1 - X&A) is plotted against t, the time in hours. Complete data for the experiments shown graphically in Figs. 1 and 2 are presented in Tables I and II according to the form of Sprinson and Rittenberg (2). Results of similar experiments on Dog 52, in agreement with data in Tables I and II, are presented in Table III. Results of calculations for the constants A and B in the empirical equa-

6 MECHANISM OF NITROGEN STORAGE. VI tion of Sprinson and Rittenberg (2), and for the rate of protein synthesis, S, and the size of the nit#rogen pool, P, as described in their paper (a), are summarized in Table IV. The data clearly show a decrease in E, and an increase in both Sk and Pk during the nitrogen storage experiments. In Table V are presented the results of calculations of the rate of amino acid catabolism, kl, and the rate at which amino acids are synthesized into TABLE Rate of Utilization of Glycine Nitrogen by Dog III hrs. O-3 3-6 6-9 9-12 12-24 24-48 SW. Dog 52, nitrogen balance atom per cent 1 excess m.e*. 0.87 0.025 0.016 1.71 0.049 0.060 1.68 0.047 0.056 1.08 0.042 0.032 1.84 0.033 0.043 7.01 0.011 0.055 o- 3 0.69 0.016 0.008 0.008 0.015 i 0.961 3-6 1.17 0.043 0.036 0.044 0.081 0.783 6-9 1.36 0.037 0.036 0.080 0.147 0.604 9-12 0.92 0.034 0.022 0.102 0.188 0.494 12-15 1.18 0.029 0.024 0.126 0.233 0.373 15-24 0.77 0.022 0.012 0.138 0.256 0.311 24-48 6.09 0.010 0.044 0.182 0.336 0.095 - Dog 52, nitrogen storage meq. 0.016 0.029 0.945 0.076 0.140 0.734 0.132 0.244 0.536 0.164 0.304 0.422 0.207 0.384 0.270 0.262 0.487 0.074 * XB = N s excreted; X0 = N s fed at zero time = 0.54 m.eq. t A = 0.526 for N balance experiment (see Table IV); A = 0.372 for N storage experiment (see Table IV). protein, kz, by the mathematical treatment of Hoberman (3). Induction of nitrogen storage resulted in a decreased rate of amino acid catabolism in all animals and an increased rate of protein synthesis. The decrease in the rate of amino acid catabolism during nitrogen storage was very marked, ranging from 42 to 67 per cent. The rate of protein synthesis during nitrogen storage was 8 to 18.5 per cent higher than that observed during nitrogen balance experiments. DISCUSSION Results of our studies on the dog during nitrogen balance and during growth hormone-induced nitrogen storage experiments show that the em-

P. D. BARTLETT AND 0. H. GAEBLER 7 pirical equation for the excretion of labeled glycine nitrogen in both the rat and the human (2) is also applicable to the dog. The rate of protein synthesis per kilo of body weight was found to average 0.62 gm. of nitrogen per day for the animals in nitrogen balance. This value lies intermediate TABLE Rate of Protein Synthesis and Size of Nitrogen Pool in Dog IV,nfii Body E* weight dog EZ 100 xa x0 o-48 hrs. Nitrogen A B S P* Ek Sk* Pk* balance kg. per Gent per cent gm. iw. gm. Am. P. 44 15.2 8.32 37.8 48.5 0.527 1.27 7.45 12.5 0.547 0.490 0.823 52 11.5 7.18 38.4 48.7 0.526 1.31 6.46 10.4 0.626 0.564 0.908 55 15.8 10.49 35.4 42.8 0.447 1.56 12.95 15.1 0.663 0.818 0.950 Average... 0.612 0.624 0.894 f0.043f0.129f0.044 Nitrogen storage 44 15.2 4.67 15.3 21.7 0.258 0.90 13.06 19.7 0.307 0.058 1.300 52 11.8 6.08 25.6 33.6 0.372 1.16 10.28 14.1 0.515 0.871 1.195 55 17.0 7.22 19.1 25.9 0.295 1.04 17.23 23.5 0.425 1.013 1.383 Average... 0.416 0.914 1.290 f0.072f0.066f0.066 * E = total nitrogen excreted during the first 24 hours of the experiment; S = gm. of nitrogen synthesized into protein per 24 hours; P = gm. of nitrogen in the metabolic pool; Ek, Sk, and Ph are the values of E, S, and P calculated on the basis of per kilo of body weight. t xe/xo is the fraction of fed Nr6 excreted in the urine. TABLE Rates of Amino Acid Catabolism and Protein Synthesis V Experimental 44 52 55 dog Nitrogen balance Nitrogen storage kl W kl kz $er cent $er hr. #er cent #er hr. per cent $er hr. per cent $er hr. 3.85 3.85 1.26 4.56 3.96 4.19 2.30 4.54 2.74 3.60 1.36 3.89 * ki = rate of amino acid catabolism. t kz = rate of transformation of amino acids into protein

8 MECHANISM OF NITROGEN STORAGE. VI between the rates of 1.0 and 0.2 gm. of nitrogen per day reported by Sprinson and Rittenberg (2) for the rat and human, respectively. Evaluation of the N15 data in terms of rate-process constants for the catabolism of amino acids into urinary nitrogen and for the synthesis of protein from amino acids according to the method of Hoberman (3) confirms the general results expressed in terms of Pk and Se values and provides in addition specific data on the rate of amino acid catabolism. In the nitrogen balance experiments lcl was found to be equal to kz in both Dogs 44 and 52. In similar studies on Dog 55 1~2 was slightly higher than ICI. During nitrogen storage experiments, the rate of protein synthesis was from 2 to 3.5 times as large as the rate of amino acid catabolism. Results of quantitative evaluation of the rate-process con&ants during nitrogen balance and nitrogen storage experiments and determination of the quantity of nitrogen synthesized into protein and the size of the nitrogen pool have confirmed, extended, and more clearly described in quantitative terms nitrogen storage induced with growth hormone in the dog. In agreement with the depressed rate of amino acid catabolism in this animal are observations of increased plasma free amino acid carboxyl nitrogen and decreased plasma glutamine amide nitrogen (12). SUMMARY 1. The rate of protein synthesis per kilo of body weight was found to average 0.62 gm. of nitrogen per day during nitrogen balance experiments on dogs. This value is intermediate between the rates of 1.0 and 0.2 gm. of nitrogen per day reported for the rat and human, respectively. The size of the nitrogen pool expressed in terms of gm. of nitrogen per kilo of body weight was increased by 44 per cent during induced nitrogen storage. 2. Amino acid catabolism was decreased from 42 to 67 per cent and the rate of protein synthesis increased from 8 to 18.5 per cent during induced nitrogen storage. 3. Amino acid catabolism and synthesis of amino acids into protein pro- ceeded at the same rate during nitrogen balance experiments. During nitrogen storage experiments, the rate of synthesis of amino acids into protein was from 2 to 3.5 times as large as the rate of amino acid catabolism. 4. The production of growth hormone-induced nitrogen storage in the adult female dog is the resultant of two over-all effects: a depressed rate of amino acid catabolism and an increased rate of protein synthesis which have been quantitatively evaluated. BIBLIOGRAPHY 1. Rittenberg, D., Cold Spring Harbor Symposia Quant. Biol., 13, 173 (1948). 2. Sprinson, D. B., and Rittenberg, D., J. Biol. Chem., 180, 715 (1949).

P. D. BARTLETT AND 0. H. GAEBLER 9 3. Hoberman, H. D., Yale J. Bid. and Med., 22, 341 (1950). 4. Hoberman, H. D., and Graff, J., Yale J. Biol. and Med., 23, 195 (1950). 5. Hoberman, H. D., J. Biol. Chem., 166, 797 (1951). 6. Bartlett, P. D., and Glynn, M., J. BioZ. Chem., 187, 253 (1950). 7. Evans, H. M., Simpson, M. E., and Li, C. H., Growth, 12, 15 (1948). 8. Li, C. H., Simpson, M. E., and Evans, H. M., Growth, 12,39 (1948). 9. Moulton, C. R., J. BioZ. Chem., 67, 79 (1923). 10. Gaebler, 0. H., Bartlett, P. D., and Sweeney, M. J., Am. J. Physiol., 166, 4% (1951). 11. Sprinson, D. B., and Rittenberg, D., J. BioZ. Chem., 160, 707 (1949). 12. Bartlett, P. D., Gaebler, 0. H., and Harmon, A., J. BioZ. Chem., 180,102l (1949).

STUDIES ON THE MECHANISM OF NITROGEN STORAGE: VI. RATE OF PROTEIN SYNTHESIS AND SIZE OF THE NITROGEN POOL Paul D. Bartlett, Oliver H. Gaebler and With the technical assistance of Beverly Cady J. Biol. Chem. 1952, 196:1-9. Access the most updated version of this article at http://www.jbc.org/content/196/1/1.citation Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/196/1/1.citation.full.html #ref-list-1