CREATININE AND CREATINE IN MUSCLE EXTRACTS.

CREATININE AND CREATINE IN MUSCLE EXTRACTS. II. THE INFLUENCE OF THE REACTION OF THE MEDIUM ON THE CREATININE-CREATINE BALANCE IN INCUBATED EXTRACTS OF MUSCLE TISSUE OF THE ALBINO RAT. BY FREDERICK S. HAMMETT. (From The Vistar Institute of dnatomy and Biology, Philadelphia.) (Received for publication, June 29, 1921.) The object, of t#his investigation was the determinat,ion of the changes that t#ake place in the creatinine and creat#ine content of extracts of muscle t#issue of the albino rat when incubated at body temperature for 24 hourswhen the reaction of the ext#ract is buffered to neutrality or alkalinity, and when the extract is unbuffered save by the Tyrode s solution used as diluent, and is allowed to develop it,s own reaction which is slightly acid to rosolic acid. The study is tjo serve as a foundation for an inquiry into the factors concerned in the creat,inine-creat#ine balance in such tissue extracts wit#h the hope t hat, some light) may be thrown on t he problems of t,he metabolism of t,hese compounds. There are t,hose who have doubted hhat t he demonstrat,ion by 8t,angassinger (I), GoMieb and Stangassinger (2, 3), Rothma,nn (4), biella,nby (5), and Myers and Fine (6) of an increase in creatinine accompanied by a decrease in creatine content of muscle tissue or muscle extract,s in ill vitro experiments is a valid indication that such a process occurs in the living organism. The objections raised to such an application of the findings have been based on evidence which has its contradictory phases, while on t#he other hand, the main facts of the strichly laborat(ory t,ests consistently point in one direct ion. The uniformity of the result s of these lat ter methods of attack can be at tributed t o t he elimination of t,he interfering factors of digestion, assimilat,ion. utilization, bacterial action, and the probable influence of organs other than the muscles on the react ion being studied. It is not necessary to go int,o the literature dealing with 135

134 Creatinine-Creatine in Muscle Extracts. II this controversy, for to those interested in the problem its main features are well known. The results of the studies of autolyzed muscle tissue or extracts have shown that the increase of the creatinine content in such preparations occurs whether the reaction of the medium is acid, neutral, or alkaline. But the data arc conflicting with respect to the relative influence of the reaction on the amount of creatine formation. The recent report of Hahn and Barkan (7) on the effects of sodium hydroxide and hydrochloric acid on this change in aqueous solutions of creatine while of interest is hardly directly comparable with the studies in which tissue extracts were used. For the purposes of this study extracts were prepared from the voluntary muscles of the hind limbs of albino rats as described in the preceding paper (8). Rats of the same sex and age were used within each series, although the sex and age differed for the different series. For each series a set of sixteen centrifuge tubes was used and into each tube 5 cc. of muscle extract. were measured, using the same pipette throughout. To the first group of four tubes there were added 4 drops of distilled water; to the second group 4 drops of Henderson s (9) phosphate mixture; and to the third 4 drops of a saturated solution of Na2HP04. The remaining four lots were used for the estimation of the preformed and total creatinine of the fresh extract. 0.5 cc. of toluene was added to the above mixtures and they were thoroughly mixed by means of a fine stream of air blown through a glass capillary dipped to the bottom of t.he tubes. When the reaction of the various groups was tested with rosolic acid, they were found to be slightly acid, neutral, and alkaline, respectively, both before and after incubation. After the contents of the tubes had been prepared as described they were incubated for 24 hours usually at a temperature of 38, although some lots were kept at 36 and 40. After incubation the preformed and total creatinine were determined according to the picric acid deproteinization method previously described (8). Parallel determinations were made and the reported values represent their averages. The statistical values of the parallel determinations are given in Table I in terms of 0.1 mm. and demonstrate that a considerable degree of reliance can be placed upon the findings.

F. S. Hammett 135 Turning now to a consideration of the results of these experiments the figures in Table II are given. They represent in mg. the amounts of total and preformed creatinine found in the fresh extracts and in the extracts after incubation under the conditions described. The percentage increase is also tabulated as are the statistical values for the series as a whole. No figures are given for creatine since they are obtained by difference and calculation and would add nothing to the argument,. It is evident that here, as with other workers, there has been an increase in the creatinine content of the extracts on incubation TABLE I. Statistical Values of the Calorimetric Readings MCSMl difference. 0.1 mm. Creatinine before incubation. 2.2 Creatinine after incubation. Acid. 2.4 Neutral 2.4 Alkaline. 3.0 Creatinine; all determinations.. 2.5 Total creatinine before incubation. 2.1 Total creatinine after incubation. Acid 2.2 Neutral. 3.6 Alkaline 2.6 Total creatinine; all determinations. 2.4 Of' the Parallel - Probable error of mean. Standard deviation. 0.1 mm. 0.1 mm. 0.25 1.76 0.31 1.98 0.31 1.84 0.38 2.24 0.15 1.97 0.25 1.80 0.39 2.03 0.49 1.94 0.46 1.91 0.18 1.94 Determinations. 0.1 mm. 0.18 whether the reaction of the medium was acid, neutral, or alkaline. This increase is statistically valid as measured by the usual criterion that the probable error of the mean must be contained in the difference between t,he means at least twice, and three times for definitely satisfactory differences. No changes in the amounts of total creatinine occur on incubation when the same standard of validity is applied. Such being the case and since there has been neither gain nor loss of total creatinine under these conditions, the increase in the creatinine must perforce have been at the expense of the creatine. Since t he muscle extracts exhibiting this phenomenon were ext,racts made with 0.22 0.22 0.27 0.11 0.18 0.28 0.35 0.32 0.13

Mean... Standard deviation Probable error of mean.. Probable error of standard deviation.... - TABLE II. Changes in the Crentinine-Great&e Balance on Incubation. Preformed creatinine. Total creatinine. Fresh. - WI. Incubated. Fresh. - Acid. Neutral. Alkaline. Acid. per cm mg. increase 0.055 0.134 143.6 0.064 0.167 161.0 0.067 0.074 10.4 0.073 0.145 98.6 0.074 0.150 102.7 0.074 0.171 131.1 0.078 0.148 89.7 0.080 0.135 68.7 0.081 0.195 140.7 0.081 0.213 163.0 0.088 0.137 55.7 0.116 0.158 36.2 -- 0.077 0.152 100.0 0.014 0.011 47.8 per cent m7. incrense per cent mg. increase w?. 0.199 261.8 0.166 201.8 5.19 0.228 256.2 0.184 187.5 5.41 0.094 40.3 0.077 14.9 3.33 0.211 189.0 0.163 123.3 5.07 0.188 154.1 0.167 125.9 4.42 0.286 286.5 0.214 189.2 5.55 0.185 137.2 0.150 92.3 4.44 0.160 100.0 0.152 90.0 4.83 0.286 253.1 0.212 161.7 5.53 0.301 271.6 0.230 184.0 5.24 0.166 88.6 0.157 78.4 5.06 0.188 62.1 0.162 39.6 4.83 0.208 175.0 0.169 124.1 4.91 0.018 85.6 0.012 59.6 0.60 mg. 5.20 5.43 3.37 4.85 4.42 5.49 4.42 4.64 5.53 4.79 4.99 4.80 er cent acrease 0.2 0.4 1.2-4.3 0.0-1.1-0.5-3.9 0.0-8.6-1.3-0.6 I Neutral. Incubated. mg. 5.20 5.43 3.33 4.98 4.52 5.46 4.50 4.64 5.53 5.28 4.93 4.83 4.83-1.5 4.88 0.57 2.6 0.58 0.003 0.002 9.1 0.004 16.7 0.002 11.6 0.12 0.11 0.5 0.11 0.002 I 0.002 I 6.6 0.003 11.8 0.002 8.2 0.08 0.08 0.4 0.08 - -- w cm1 LfYease 0.2 0.4 0.0-1.8 2.3-1.6 1.4-3.9 0.0 0.8-2.6 0.0-0.4 1.7 0.3 0.2 T -I - Alkaline mg. 5.22 0.6 5.43 0.4 3.33 0.0 5.05-0.4 4.42 0.0 5.48-1.2 4.45 0.2 4.62-4.4 5.49-0.7 5.29 1.0 4.93-2.6 4.80-0.6 4.88-0.6 0.59 1.4 0.12 0.3 0.08 0.2-7

F. S. Hammett 137 Tyrode s solution which simulates to a considerable degree the medium in which the reactions of the living organism take place, I am of the opinion that we are justified in assuming, until it has been disproved by critical experiment, that there occurs in the muscles of the living organism a formation of creatinine from the muscle creatine and that the endogenous source of the urinary creatinine is the muscle creatine. This lack of destruction of total creatinine just discussed confirms the findings of Mellanby (5) and Myers and Fine (6) and fails to substantiate the results of Gottlieb and Stangassinger (2). Experiments where putrefaction was allowed to occur, and which will be presented presently, tell another story. Now when the percentage increase in creatinine is considered it, is seen that this increase is regulated in part by the reaction of the medium, for it is least in the acid solutions, greatest in the neutral solutions, and between the two in the alkaline soluhions. This relationship is consistently constant in all of the twelve experiments reported and is substantiated by the statistical calculations. It is not in agreement with the result of Rothmann (4), Myers and Fine (6), or Hahn and Barkan (7). For the two former found an apparent acceleration of the reaction by acid and the latter that alkali retarded the change of creatinine to creatine as compared with acid. The studies of Hahn and Barkan (7), however, are hardly comparable with the studies made with tissue extracts. When one looks at the results of Myers and Fine (6) given in Table VII of their paper, it is seen that when the autolyzing mixtures were buffered to neutrality by phosphate mixture a somewhat greater creatinine formation took place than when the tissue was treated with water alone. My results confirm this finding in principle. Nevertheless the studies of Myers and Fine (6) are not, strictly comparable with mine inasmuch as they used whole muscle tissue, their periods of autolysis were extended over a longer period and they used an acid not normally found in muscle tissue. Such being the case it is evident that a slightly acid or an alkaline reaction retards the transformation of creatine to creatinine in muscle extracts when incubated for 24 hours at body temperature. This transformation occurs at a maximum when the reaction of the digesting mixtures is buffered to neutrality by a

138 Creatinine-Creatine in Muscle Extracts. II phosphate mixture. These facts serve as a partial explanation of the observations of Underhill (10) that creatinuria is frequently an accompaniment of induced acidosis and of Underhill and Baumann (11) that a marked increased creatine excretion may be found in experimental alkalosis, and other apparently anomalous results of the studies of creatinuria, if we admit that the urinary creatinine is largely derived from the muscle creatine, and in spite of the opinions of Denis and Minot (12) and Gamble and Goldschmidt (13) that the acid-base equilibrium has nothing to do with the condition. For since it is shown that both a slight acidity and an alkalinity retard the transformation of creatine to creatinine in muscle extracts it is possible to consider that if similar tendencies are present in the living organism, even though fleeting, they may give rise to similar effects, and if that phase of muscle metabolism which results in creatine formation continues at the same or even a diminished rate, there is produced a relatively greater concentration of creatine in the circulation, part of which at least finds its way to the kidneys and is excreted. That such an increase in blood creatine can occur and continue for days under changed conditions of muscular activity I have already demonstrated (14), though this phase of t,he problem is not, necessarily at present connected with the question of creatinuria and acid-base equilibrium. Since &se experiments demonstrate conclusively that there is no loss of total creatinine on incubation under sterile conditions the figures given in Table III are particularly interesting, from the point of view of the contention of Gottlieb and Stangassinger (3) of the presence in muscle tissue of creatinine-and creatinedestroying enzymes. The results in this table were obtained from extracts which had been allowed to undergo putrefaction during the incubation. It will be seen that there has occurred a marked loss of total creatinine that is statistically valid. This supports the findings of Mellanby (5) that only when bacterial decomposition occurs does there take place a destruction of creatine or creatinine, and plainly shows the cause of the results reported by the proponents of the creatinase and creatase theory. However, it is quite probable that the transformation of creatine to creatinine in muscle extracts is brought about by an enzyme, in view of the fact that t.he change occurs in the

TABLE III. Mean...... Standard deviation Probable error of mean... Probable error of standard deviation........... Effect of Putrefaction on Changes in Creatinine-Creatine Balance During Incubation. - 1 Fresh. VW. 0.069 0.104 0.118 0.121 0.103 0.021 0.007 0.005 Preformed rreatinine.,t Q. Incubated. Acid. Neutral. Alkaline. per cent er cent per cent mj. increase Fresh. increase mq. increase mo. 0.500 624.9 0.540 682.8 0.509 637.7 5.55 0.544 422.9 0.588 465.5 0.653 527.9 4.48 0.300 154.2 0.273 131.4 0.632 435.5 5.36 0.588 386.0 0.741 512.3 0.690 470.1 5.30 -- 0.483 396.0 0.535 448.0 0.621 517.8 5.17 0.111 167.2 0.169 200.1 0.068 76.7 0.41 0.037 56.4 0.057 67.5 0.023 26.0 0.14 1.46 0.65 0.21 0.026 39.8 0.040 47.6 0.016 18.3 0.10 0.16 mo. 1.99 0.57 2.15 1.12 Total creatinine Incubated Acid. 1 Neutral. 1 Alkaline. er cent ncrease mg. -64.1 3.00-87.3-59.9 5.36-78.8 1.18-72.5 3.18 11.2 1.71 3.8 0.58 2.8 0.41 er cent nertxme mo. -45.9 1.28 0.83 0.0 2.65-77.2 0.96-40.8 1.43 31.4 0.72 10.6 0.24 /I W cent ncrease r -76.9 r;ll -72.7 3 16.7 5.7 - /

140 Creatinine-Creatine in Muscle Extracts. II neutral buffered solutions at a much greater rate than in either the acid or alkaline solutions of muscle extracts or aqueous solutions, or in aqueous solutions uncatalyxed by acid or alkali. It is interesting to note that the increase of creatinine in these putrefying extracts is much greater than is that which occurs in sterile mixtures. Whether this is a true increase or whether other products are produced by the bacterial action which give the color test for creatinine I am unable to state. The great,est increase takes place in these solutions whose reaction is alkaline. It is also interesting to note that the destruction of the total creatinine occurs about equally well in acid or alkaline media while it is much less in the solutions buffered to neutrality. This may be a direct destruction of creatine or the creatine may first be changed to creatinine which is then destroyed. An explanation of these phenomena is beyond the scope of the present paper, however. The main fact to be gathered is that the only time a destruction of total creatinine is demonstrable is when putrefaction occurs in the incubating extracts. It should be noted in conclusion that slight differences in temperature during incubation result in differences in amounts of creatinine formation, in that at the lower temperatures the transformation was less. This confirms Myers and Fine (6). SUMMARY AND CONCLUSIONS. When extracts of muscle tissue of the albino rat are incubated at body temperature for 24 hours there occurs an increase in the creatinine content the relative degree of which depends in part upon the reaction of the incubated extract. When the extract is allowed to develop its own reaction, which is slightly acid to rosolic acid, an increase of 100 per cent takes place. When the extract is buffered to neutrality by phosphate mixture the increase is 175 per cent, and when the extract is made slightly alkaline the increase is 124 per cent. Since there is no change in the total creatinine content of these extracts t,his increase in creat,inine must take place at the expense of the creatine present. Moreover, since the conditions of the experiments simulate to a considerable degree condit,ions in the living tissue in that the reactions took place in muscle extract diluted with Tyrode s solution it is probable that creatinine is formed from creatine in muscle tissue in the living

F. S. Hammett organism. The apparent anomaly of an increased creatine excretion in conditions of experimental acidosis and alkalosis is explicable in part on the basis of the retardation of creatinine formation from creatine in incubated muscle extracts when the reaction is slightly acid or alkaline. If similar effects are produced in the organism the continued production of creatine as a result of a phase of muscle metabolism would result in a relatively greater concentration of this in the blood and its excretion in larger amounts in the urine. It is probable that the transformation of creatine to creatinine in the muscle extracts is facilitated by an enzyme, but no evidence is afforded of the presence in such extracts of any creatinase or Lcreatase. The only time when a destruction of creatinine or creatine takes place is when the extracts undergo putrefaction. BIBLIOGRAPHY. 1. Stangassinger, R., 2. physiol. Chem., 1908, Iv, 295. 2. Gottlieb, R., and Stangassinger, R., 2. physiol. Chem., 1905, Iv, 322. 3. Gottlieb, R., and Stangassinger, R., 8. physiol. Chem., 1907, Iii, 1. 4. Rothmann, A., Z. physiol. Chem., 1908, lvii, 131. 5. Mellanby, E., J. Physiol., 1907-08, xxxvi, 447. 6. Myers, V. C., and Fine, M. S., J. Biol. Chem., 1915, xxi, 583. 7. Hahn, A., atid Barkan, G., 2. Biol., 1920, lxxii, 25. 8. Hammett, F. S., J. BioZ. Chem., 1921, xlviii, 127. 9. Henderson, L. J., Am. J. Physiol., 1905, xv, 257. 10. Underhill, F. P., J. Biol. Chem., 1916, xxvii, 127. 11. Underhill, F. P., and Baumann, E. J., J. BioZ. Chem., 1916, xxvii, 151. 12. Denis, W., and Minot, A. S., J. BioZ. Chem., 1919, xxxvii, 245. 13. Gamble, J. L., and Goldschmidt, S., J. Biol. Chem., 1919, xl, 199. 14. Hammett, F. S., J. Am. Med. Assn., 1921, Ixxvi, 502.

CREATININE AND CREATINE IN MUSCLE EXTRACTS: II. THE INFLUENCE OF THE REACTION OF THE MEDIUM ON THE CREATININE-CREATINE BALANCE IN INCUBATED EXTRACTS OF MUSCLE TISSUE OF THE ALBINO RAT Frederick S. Hammett J. Biol. Chem. 1921, 48:133-141. Access the most updated version of this article at http://www.jbc.org/content/48/1/133.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/48/1/133.citation.full.ht ml#ref-list-1