6I unstable compound with phosphoric acid, which disappears during fatigue. (From the Department of Physiology, The University, Sheffield.

Transcription

1 6I2.744 ON THE CREATINE AND PHOSPHORUS CONTENT OF MUSCLE. BY MARION BROWN1 AND C. G. IMRIE. (From the Department of Physiology, The University, Sheffield.) IT is well known that the concentration of creatine in the skeletal muscles of mammals (dog, cat, rabbit) may be increased by the oral, subcutaneous or intravenous administration of this substance [F olin and D e nis, 1912; Myers and Fine, 1913 a]. The increment in the muscle creatine recorded by these observers varies from 5 to 27 p.c., except in one experiment of Folin's [1912], where an increase of 70 p.c. was found in the muscles of a cat following the absorption of creatine from a loop of its intestine. Since no quantitative relationship appeared to exist between the amount administered and that taken up by the muscles in these experiments, it seems likely that the capacity of the muscles to take up creatine must be influenced by some factor or factors other than its concentration in the blood or body fluids. At the time when the experiments referred to above were performed, nothing was known regarding the chemical combination in which creatine exists in the muscle, but since the work of Fiske and Subbarow [1927] and G. P. and P. Eggleton [1927], it appears that the greater proportion, if not all, of the creatine in the striated muscles is present as an unstable compound with phosphoric acid, which disappears during fatigue and reappears at some stage in the recovery process. These observations suggested to us the possibility of the phosphoric acid exercising a controlling influence on the capacity of the skeletal muscle to take up creatine, and in order to test this hypothesis the concentration of phosphoric acid and of creatine in the muscles of cats has been determined under certain experimental conditions which included the administration of creatine and of phosphates. METHODS. The phosphorus was determined by the E gglet on s' [1927] modification of Brigg's method. The muscles were freed from fascia, in situ, care 1 Ne'e Marion Hirst.

2 CREATINE AND PHOSPHORUS IN MUSCLE. being taken to avoid stimulation. Pieces of muscle, about 400 mg., were removed, rapidly weighed on a torsion balance and immersed in liquid air until frozen. Subsequent manipulations were carried out at approximately 00 C.; the dishes and reagents employed were surrounded by a freezing mixture. The frozen tissue was crushed in a mortar and extracted with 5 p.c. trichloro-acetic acid. The colorimetric procedure was carried out on the filtered extract; phosphate solutions of appropriate strengths were used as standards. Readings were made 2, 4, 6, 8 and 50 minutes after the addition of the reagents to the extract. The values obtained for the first four readings plotted against time on a graph fall on a straight line. The value for zero time was obtained by extrapolation. This value gives the free inorganic phosphorus, that at the end of 50 minutes the total phosphorus' and the difference between these values the labile phosphorus (phosphagen). The creatine in the muscle was determined by Folin's method [1914], but smaller amounts of muscle were used: the water content by drying to constant weight in a desiccator in vacuo. Cats anesthetized with chloralose were used. Ansesthesia was first induced with ether, followed by intravenous injections of chloralose, 5 c.c. of a saturated solution per kg. of body weight. In a few instances it was necessary to give additional chloralose during the course of the experiment. RESULTS. Determinations as described above have been made on thirty-one muscles removed from twenty cats. They represent in the main control observations carried out at the beginning of the experiments about to be described. The results are set out in Table I. The results are arranged in order of decreasing values of creatine. Certain points arise on which comment may be made. The amount of creatine in the muscles of cats varies widely, between 603 and 402 mg., the average for the whole series being 531 mg. per 100 g. of moist tissue. Variations of a similar order have been reported previously [Pekelharing and van Hoogenhuyze, 1910; Myers and Fine, 1913 b; Folin and Buckman, 1914; Eagles, 1928] and others. Not only does the creatine content of the same muscles in different cats vary, but considerable variations exist in the different muscles of the same cat. This has been observed previously by the investigators to whom reference has just been made. According to Mellanby [1908] the concentration 1 By total phosphorus is meant the total acid-soluble phosphorus. 215

3 216 Creatine GROUP 1: Averages 551 GROUP 2: Averages 463 Difference + 19 (p.c.) M. BROWN AND C. G. IMRIE. Total phosphorus 97*8 78*8 80* * *1 84*5 94*0 89* * * *4 83* *9 87* *0 +8 TABLE I. Inorganic phosphorus *0 17*0 21* * *0 24*5 22*5 15*0 17*0 25*0 15* *0 26*5 21* *0 32*0 26*5 25*0 21*0 19*0 30* * Labile phosphorus 69* * *1 72* * * * *0 68* * Water content; (p.c.) * *2 Creatine and phosphorus are given in mg./100 g. moist tissue. 1 In all tables by total phosphorus is meant total acid-soluble phosphorus. 71*7 74*1 72*6 74* *6 72' * *0 71* of creatine in the muscles of newly born kittens is very low, 177 mg. per 100 g., and Myers and Fine [1913 b] found that the amount of creatine in the muscles of kittens increased during the first seven weeks of life, when the concentration of the adult muscle was attained. The cats used in our experiments were all older than this, so that age does not account for the differences observed. The amount of "total phosphorus" contained in the muscle also varies over a fairly wide range, from to 71-6 mg. per 100 g. The inorganic phosphorus varies more widely still, and such variations must be reflected in the labile phosphorus, since this value is derived from the difference between the total and inorganic phosphorus.

4 CREATINE AND PHOSPHORUS IN MUSCLE. In Table I, with few exceptions, high values for creatine tend to go along with high values for total phosphorus, the average for the first group being 19 and 8 p.c. respectively higher than those of the second. As the average values for the inorganic phosphorus in the two groups differ in the opposite sense, those for labile phosphorus are lower in the second group than in the first by more than those for the total phosphorus, 9 mg./100 g. as compared with 6-8 mg./100 g. If in phosphagenl there is one atom of phosphorus to one molecule of creatine, since Group 1 contains 9 mg. more labile phosphorus than Group 2, the corresponding increase in creatine would be 38 mg.; the actual increase is 88 mg./100 g. As there are 6*8 mg. more total phosphorus in Group 1, the corresponding amount of creatine would be about 28 mg. TABLE II. Control experiments, cats, chloralose. Total Inorganic Labile Time phos- phos- phos- (P.M.) Creatine phorus phorus phorus Exp. xxviii: Water content Muscles L. rectus femoris 4* R. rectus femoris L. tibialis anticus *7 21* R. tibialis anticus Maximal variation (p.c.) 7x O5 Exp. xi: x9 19X X1 L. rectus femoris '8 R. rectus femoris L. tibiajis anticus Maximal varia * tion (p.c.) The im a ment. variation is expressed as a percentage of the lowest value in each experi- Table II gives the results of two control experiments in which different muscles of the same cat were examined at intervals varying from 4I to 2 hours in order to ascertain the degree of variations in the creatine and phosphorus content under such conditions. Differences in the concentration of these constituents do exist; the maximal variation in the creatine is 7.7 p.c., that of the total phosphorus 8-7 p.c. Differences of a higher order appear in the inorganic phosphorus, and this naturally affects the values for the labile phosphorus. On four occasions, 200 mg. creatine per kg. of body weight were injected intravenously in a concentration of 1 p.c. in normal saline. Muscles were removed for examination before and at varying intervals PH. LXXI

5 218 M. BROWN AND C. G. IMRIE. of time after the injection. The figures for these experiments which are shown in Table III are inconclusive. In one instance, Exp. ii, the concentration of creatine in a muscle 2 hours following the injection is actually less than in a muscle removed before the injection was begun, whereas in Exp. iii, an increase of approximately 17 p.c. occurred. The concentration of total phosphorus presents certain variations, but of an order no greater than in the control experiments. Similar comments may be made regarding the inorganic and labile phosphorus. TABLE III. Before and after intravenous injections of creatine. Total Inorganic Labile Water phos- phos- phos- content Exp. Time Creatine phorus phorus phorus (p.c.) iv Before hr. later * Variation (p.c.) ii Before hr. later Variation (p.c.) *1 iii Before... 3 hr. later Variation (p.c.) vi Before hr. later... 4 hr. later * * *9 30* * Variation (p.c.) Muscles were removed in the following order, left rectus femoris, right rectus femoris and left tibialis anticus (in Exp. vi). It has been observed that an increase in the muscle creatine does not invariably follow the injection of creatine into the veins of a cat. Folin and Denis [1912] obtained more consistent results when the creatine was introduced into the duodenum after the renal arteries were ligated in order to prevent the excretion of creatine by the kidney. This procedure was adopted therefore in other experiments. In six cats under chloralose the renal arteries were ligated but no creatine given. Pieces of different muscles removed for examination at intervals show no regular progressive increase in concentration of creatine and phosphorus. The variations are similar to those in Table II when reckoned in a similar way. In Table V are set out the results of five experiments where, after ligation of the renal arteries, creatine in large amounts (3 g.) was introduced into the duodenum. In one of these acid potassium phosphate was

6 CREATI-NE AND PHOSPHORUS IN MUSCLE. 219 TABLE IV. Control experiments, cats, chloralose, ligation of the renal arteries, no creatine given. Total Inorganic Labile Water phos- phos- phos- content Exp. Time Creatine phorus phorus phorus (p.c.) xxi Maximal variation (p.c.) xxii Maximal variation (p.c.) xxiii Maximal variation (p.c.) xxiv Maximal variation (p.c.) xxv Maximal va riation (p.c.) xxvi Maximal variation (p.c.) Average of percentage maximal variation: Creatine 4-6 Total phosphorus 11-2 Muscles were removed in the following order, left rectus femoris, right rectus femoris, left tibialis anticus and right tibialis anticus. given as well; the dose, however, was too small to effect the concentration of phosphorus appreciably. Larger doses proved to be toxic, presumably due to the potassium. The figures show quite clearly that the changes in the concentration of creatine and phosphorus are not only of a higher order than in the controls, but the concentrations appear to increase progressively throughout the experiments, whereas in the controls the variations were somewhat irregular. The average percentage increase for the creatine is 11-6, 15-2

7 220 M. BROWN AND C. G. IMRIE. for the phosphorus 17-2, an increase of 7 and 6 respectively above the maximal variations in the controls. TABLE V. Cats, chloralose, renal arteries ligated, creatine introduced into the duodenum. Total Inorganic Labile Water phos- phos- phos- content Exp. Time Creatine phorus phorus phorus (p.c.) xiii * Maximal variation (p.c.) 23-2 xiv Maximal variation (p.c.) xv Maximal variation (p.c.) xvi Maximal variation (p.c.) xix Maximal variation (p.c.) Average of percentage increase: Creatine 11-6 Total phosphorus 17-2 Average of percentage maximal variation (Table IV): Creatine 4-6 Total phosphorus 11-2 Average of percentage increase above maximal varia. Creatine 7-0 tion in the control experiments: Total phosphorus 6-0 The muscles were removed in the same order as in the preceding table. CONCLUSIONS. 1. In the muscles of normal cats when the amount of creatine is high there is a tendency for the concentration of total acid-soluble phosphorus and the proportion of it present as phosphagen also to be high; although this is not constantly the case, as if a certain portion of the creatine in muscle were present not as phosphagen. 2. When the concentration of creatine in the muscles is increased following the absorption of creatine introduced into the duodenum, there

8 CREATINE AND PHOSPHORUS IN MUSCLE. 221 is evidence that the concentration of total acid-soluble phosphorus is also increased. It is a pleasure to acknowledge our indebtedness to Prof. Leathes for his interest and advice. The expenses of this research were defrayed by the Medical Research Council. REFERENCES. Eagles, B. A. (1928). Creatine and Creatinine, p. 75. Monographs on Biochemistry. A. Hunter. Eggleton, G. P. and Eggleton, P. (1927). Biochem. J. 21, 190. Fiske, C. H. and Subbarow, Y. (1927). Science, N. York, 45, 401. Folin, 0. (1914). J. Biol. Chem. 17, 481. Folin, 0. and Buckman, T. E. (1914). J. Biol. Chem. 17, 483. Folin, 0. and Denis, W. (1912). J. Biol. Chem. 12, 141. Folin, 0. and Denis, W. (1914). J. Biol. Chem. 17, 493. Mellanby, E. (1908). J. Phy8iol. 36, 447. Myers, V. C. and Fine, M. S. (1913 a). J. Biol. Chem. 16, 169. Myers, V. C. and Fine, M. S. (1913 b). J. Biol. Chem. 14, 9. Pekelharing, C. A. and van Hoogenhuyze, C. J. C. (1910). Z. Phy8iol. Chem. 64, 262.