Arginin. BY W. H. THOMPSON, M.D., Sc.D.

Transcription

1 THE PHYSIOLOGICAL EFFECTS OF PEPTONE AND ALLIED PRODUCTS. PART VI. The Metabolism of Arginin. BY W. H. THOMPSON, M.D., Sc.D. CONTENTS. Methods..p The effects of Arginin Feeding... p Results of Hypodermic Injection of Arginin.. p The Urea-Nitrogen Quotient during Arginin Metabolismn p The Nitrogen of the Fnces...p Hexone Bases and Glycosiia...p Conclusions..p Protocols..p (OF the following experimeilts oine was performied in the Pharmakologisches Institut, Marburg, one in the Physiological Laboratory, Queen's College, Belfast, and one in the Physiological Department, Trinity College, Dublin. To Prof. A. Kossel I am much indebted for the substance employed in all and for valuable assistanice in the work; to Pi-of. H. Meyer for kind permnission) to coniduct one of the experimients in his laboratory.) In previous experiments I had ascertained that arginin, in the form of a neutral solution of its carbonate, rnay be injected into the circulation of a dog without producing harmnful, or indeed any pronounced effects'. It proved inert as regards blood pressure and had no immrlediate influence on urinary secretion, beyond that which could be attributed to the amount of fluid injected. In the following experiments arginin was administered to dogs, both hypodermically and with their food, the object being to observe in what form it reappeared in the excreta, and also what effects it might produce oni nitrogenous metabolism. When suibmitted to the action of barium hydrate, arginin, as is well known, readily undergoes hydrolytic cleavage, yielding urea an(d ornithin (diamido-valerianic acid), half of the arginin nitrogen appearing as urea and half as ornithin. Or by weight from 192 parts arginin and water, I Zeitschr. f. physiol. Chemnie, xxix. s

2 138 W. H. THOMPSON. 60 parts appear as uirea anid 132 parts as ornithin. It was to be expected therefore that if metabolised in the animal body, at least part would be transformed into urea and be excreted as such in the urine. In carrying out the experiments, the animals were brought into nitrogenous equilibrium. The diet consisted of lean minced beef, dried bread crumb, and lard. Arginin was then added to the food for one or more days. After this, the former diet was continued and when the output of nitrogen hlad returned approximately to its previous level, the substance was given by hypodermic in,jection. Each animal was thus enmployed for a feeding and a hypodermic experiment. In all, three experiments of each kind were carried out. In two cases the arginin feeding preceded the hypodermic injection, in one the reverse order was followed. THE EFFECTS OF ARGININ FEEDING. In two of the following experiments the arginin carbonate was dissolved in physiological solution of sodium chloride, the mixture then neutralised with hydrochloric acid and added to the food. In experiment 3, the arginin carbonate was mixed as such with the food, and given on three successive days. The following is a short summary of the results in each case. Column I. gives the average output of nitrogen in grammes, before the admirnistration, column II. duiring feeding period, axnd columnn II1. the same after the arginin was stopped. Columin IV. gives the nitrogen equivalent of the arginin administered. The following table gives a short summary of the results in each case. In column I. the average output of nitrogen in the urine before the administration is given (a) as total nitrogen, (b) as urea. Columns II. and III. give the same for the feeding period and after-period respectively. Column IV. represents the nitrogen-equivalent of the arginin administered. TABLE A. Showing the effects of Arginin feeding. I. (Before) II. (During) III. (After) IV. Dose given a b a b a b Total N Urea-N Total N Urea-N Total N Urea-N Arginin-N Exp e Exp Exp , per day for 3 days. N.B. In column I. the figures for Exp. 1 represent averages of 4 days, for Exp. 2 averages of 3 days, for Exp. 3, averages of 6 days. In column II. Exp. 3 the figures are averages of 3 days.

3 A RGININ METABOLISM. 139 It will be seen that in all three experiments there was a marked increase in the excretion of urinary nitrogen during the feeding period, and that this did not subside immediately after the arginin was stopped. As will be seen also the greater part of the increase was due to a formation of urea. In Exps. 1 and 2 the arginin was given for one day only. In the first of these the increased output of nitrogen for the day of feeding and day after amounted to grin. Of this grmn. appeared as urea, while the total arginin-n administered was grm. That is to say, within the period of observation /0 of the arginin nitrogen came out in the urine as urea. In Exp. 2 the output of total nitrogen for the two days was increased by grm. Of this 0'612 grm. came ouit as urea, the nitrogen of the arginin administered being 084 grm. Thus /o of the latter reappeared in the urine as urea. In Exp. 3 the feeding period extended over three days, and the figures given for this experiment in column II. represent the mean output for this period. Here also the nitrogen output remained distinctly above the normal for one day after the arginin was stopped. During the three days of feeding, there was a total excess of nitrogen amounting to grms. to which must be added grm. for the day following, making in all grms. The urea excess for the feeding period amouinted to grms. and for the day after grm., in all grms. The total arginin administered during the three days contained grms. More total nitrogen was therefore excreted in the observation period than could have come fromi the arginin, while 96'31 0/0 of the arginin nitrogen reappeared as urea. Hence it must be concluded that the substance, in addition to forming urea, has a stimulatirng effect upon proteid metabolism. The animals' weibht also slightly declined during the feeding period. In two of the experiments observations were made on the excretion phosphates. These showed a moderate increase during the administration of arginin in both cases. Thus in Exp. 2 the average daily output previous to the feeding was 0 :395 grm. total phosphates. On the day of feeding this rose to 0'43.50 grm. In Exp. 3 the corresponding figures were 0'4737 and grm. respectively. The urine was not analysed for the presence of ornitbin. It is hardly conceivable from the foregoing results that this substance could have been present in appreciable amount. These distinctly indicate that a mnuch larger proportion of arginin is ultimately transformed into

4 140 W. H. THOMPSON. urea in the animal body than has been obtained by hydrolytic cleavage in the laboratory. Since the foregoing experiments were carried out, Kossel and D akin1 have succeeded in extracting a ferment from the liver wbich splits arginin into urea and ornithin. The highest proportion of urea obtained by them amounted to 400/0 by weight of the arginin employed, the digestion having been allowed to continue for twelve days. Comparing these results with my own, it seems probable that in the metabolism of arginin in the animal body the ornithin residue is likewise converted in large part into urea. RESULTS OF HYPODERMIC INJECTION OF ARGININ. The following table gives a short summary of the effects produced by hypodermic injection of arginin solutions. In all cases the carbonate was emriployed and the solution almost neutralised with hydrochloric acid. The precaution of sterilising the solution before injection was also observed. The table is constructed in the same way as table A. Column I. represents the excretion of total urinary nitrogen and of urea nitrogen before the injection of arginin. Coluimns II. and III. give the same for the day of injection and day after. Column IV. gives tile nitrogen of the arginin injected. TABLE B. S/owiung the efects of subcutaneous injection of Arginin. I. (Before) II. (Injection) III. (After) IV. a b a b a b Total N Urea-N Total N Urea-N Total N Urea-N Arginin-N Exp , P1648 Exp s X Exp , N.B. In column I. the figures for Exp. 1 represent averages of 4 days, for Exp. 2 averages of 3 days before any arginin was administered. Those for Exp. 3 are averages of 2 days, after the feeding, and immediately preceding the injection. The hypodermic injection of arginin in all three experimnents gave rise to a nmarked increase in the excretion of urinary nitrogen on the day of experiment. This was continued to a much less degree on the day following. In Exp. 1 the excess of nitrogen for the two days amounted to grms., of which urea accounted for grms. In this case the arginin given contained grms. nitrogen. In Exp. 2 the total nitrogen excess for the same period was 1292 grins. and of 1 Kossel and Dakin. Zeitschr. f. physiol. Chemie, XLI., p

5 ARGININ META BOLISM. urea nitrogen 0941 grm. The arginini given contained 084 grm. nitrogen. The corresponding quantities for Exp. 3 were: total N excess grm., urea excess grm. nitrogeni, arginin given grm. nitrogen. In Exps. 1 and 2 more uirea was excreted than could have been (Jerived from the arginin. employed. From the figures given in the table this does not seem to have been the case in Exp. 3. But in this experimient the output of nitrogen remained above the normal for two days instead of one, after the injection. If this be taken account of, then the excess of total nitrogen for the three days (including day of experiment) amounts to 0)880O5 grm. and of uirea to grm. as against anl injection of grm. of arginin nitrogen. It is I think quite legitinmate in Exp. 3 to make this inclusion, because, apart from the fact that dogs may react differently in regard to the rate with which they excrete a substance (such as arginin) administered hypodermically, it should be mentioned that the injection in this case was made later on the day of experiment than in the previous cases. Apparently therefore, with hypodermic injection of arginin as with feeding, practically the whole of the stubstance is converted into urea in the animal body. The results also support the hypothesis that arginin acts as a metabolic stimulant. It seemed not unlikely however that the effects of the solvent employed might also account in part for the output of nitrogen beyond that containied in the arginin injected. Control experiments were therefore performed on two of the dogs, namely those employed in Exps. 2 and 3. In one of these dogs, the effects in this respect of 0*90/, NaCI and also of 1 0/0 sodium acetate were observed. lu the other the effects of 0'9 0/0 NaCl. The following table shows the results, columns I. II. and III. giving the same values as in previous tables. The quantity of fluid injected in each case was 25 c.c. TABLE C. Showing the effects of Hypodermic Injections of salt solutions upon the excretion of Urinary Nitrogen. I. II. III. Total N Urea-N Total N Urea-N Total N Urea-N Exp. 2 (a). NaCl 2'5920 1'9160 2' Exp. 2 (b). NaC2H3O2 2'6320 1' * Exp. 3. NaCl '968 2'632 2' Sodium acetate, in the single case in which it was employed, caused a marked rise in the excretion of urinary nitrogen. But this result hardly bears upon the present investigation. The effects of sodiuim

6 142 W. H. THOMPSON. chloride solution were much less marked and differed in the two experiments. In the one a slight increase was caused, in the other a very slight effect in the opposite direction. The solvent per se in the quantities used cannot therefore be held to account for the excess of nitrogen output above that contained in the arginin employed. The conclusion consequently seems fully justified that this excess must be due to the fact that arginin distinctly promotes nitrogenous metabolism both when hypodermically injected, and also to a less degree when given per os. THE UREA-NITROGEN QUOTIENT. Assuming that arginin in the animal body is mainly converted into urea one would expect that when administered as in the foregoing experiments, the proportion which the urea bears to the total nitrogen of the urine would be relatively increased. Accordingly this proportion was calculated for each of the experiments: (a) when the animal was on normal diet, (b) when arginin was added thereto, and (c) when arginin was injected under the skin. The following table gives the percentage of urea nitrogen to total nitrogen in each case: Normal Feeding Injection Exp A Exp , Exp On the whole there was therefore a slight rise in the urea quotient during the administration of arginin. This applies to all of the feeding experimenits and also to two of the injection experiments. The injection in Exp. 3 appears to be an exception, but it should be borne in mind that in this case, as explained before, the whole of the arginin did not come out in the urine of the day of experitnent. More of it remained to be excreted on the following day than in previous experiments, and this has not been taken account of in the above calculations. THE NITROGEN OF THE FAECES. In Exp. 3 an analysis of the faeces was carried out to determine whether any appreciable quantity of the arginin escaped absorption in the alimentary canal. The faeces of four periods were collected and

7 ARGININ METABOLISM. treated in the recognised way. The first period was that before the administration of arginin, the second when the substance was given with the food, the third included the day of subcutaneous injection, and the fourth the remaining period of the experiment. The average daily excretion of nitrogen in the feeces was; during the first period, grm. during the feeding period, 0A409 grm. during the third period, grin. during the last period, 0426 grm. It would appear therefore that the absorption of nitrogenous food from the alimentary canal was even more complete during the administration of arginin than under normal conditions. Consequiently it can hardly be supposed that any of the substance escaped with the feces. Analyses of the faeces in Exps. 1 and 2 were not carried out. Addendum. HEXONE BASES AND GLYCOSURIA. 143 It has been asserted by Muller and Seeman' as well as by other investigators that the sugar which appears in the urine of diabetic patients on a purely proteid diet must be derived through the hexone bases or other six carbon derivates of proteids, and Cohn2 claims that feeding rabbits with leucin increases to some extent the glycogen in their livers. Halsey3 did not find however that feeding with leucin increased the sugar of the urine in diabetes, or if so only indirectly. In the first of the arginin experiments here recorded the urine after hypodermic injection when examined with the polariscope was found to have a dextrorotatory action. In a tube of 4 cm. length the mean of six readings gave a rotation of It was assumed that the optically active substance was arginin, and on calculating the amount contained in the urine of the day (made up to 500 c.c.), [ad] being taken as (Lawrow), this appeared to be 3 75 grms. It seemed therefore that more than half of the arginin injected was excreted unaltered in the urine. An attempt was consequently made to recover the substance, the greater part of the day's secretion of urine being employed for this purpose. No arginin was discovered however and the remainder of the urine was 1 Deutsche med. Wochenschr. 1899, s Zeitschr. f. physiol. Chemie, xxviii Sitzungsber. d. Gesellsch. d. Natwissensch. z. Marburg, 1899, s. Journ. of Physiol. x. p ; also Americ. PH. XXXII. 10

8 144 W. H. THOMPSON. examined for other dextrorotatorv substances. In the end it came out that the optically active suibstance proved to be dextrose. Reduction was obtained with Trommer's test, osazone crystals were formed with phenyl hydrazin and acetic acid, and finally fermentation with yeast gave positive results. In one of my early experiments with arginin in which the substance was directly injected as carbonate into the circulation, and the urine collected from the ureters, the urine likewise gave a dextrorotator.y effect. The rotation amounted to in a tube of 1 cm. length. This was taken at the time to mean an excretion of part of the arginin unchanged, but the occurrence of dextrose was not then suspected and no steps were taken to exclude this possible source of error. I recently repeated the experiment, and took the precaution of fermenting the urine with yeast before examining with the polarimeter. The result showed that the urine was optically inactive. In both these latter experiments the animals were anaesthetised with ether-chloroform mixture, which of itself would account for the presence of some dextrose in the urine. The importance of the discovery of dextrose in the urine of the first hypodermic and feeding experiinents, where the influence of an anassthetic could not have come in, led me to carefully examine the urines in both the other similar experiments, but in neither was sugar discovered. At most, therefore, one can only say that apparently arginin in some cases, when administered hypodermically or with the food, produces glycosuria. It must be borne in mind however that even for the one case recorded the proof of arginin being the direct cause of the glycosuria is not absolutely complete. It may have been that the solvent alone, if injected subcutaneously in this particular animal, would have had a like effect, or it may even have been that the animal had already suffered from slight glycosuria, before the experiment was undertaken. Neither possibility was definitely excluded. CONCLUSIONS. 1. Feeding with arginin as chloride or carbonate gives rise to an increase of nitrogen in the urine, the greater part of which appears as urea. 2. For every 100 parts nitrogen administered as arginin, the amount which reappeared in the form of urea varied between 72-8 and 963 0/0. When transformed in the laboratory only half the nitrogen appears as urea, the other half as ornithin.

9 ARGININ METABOLISM In the animal body either ornithin is not formed, or if formed is reconverted for the most part into urea. 4. Much the same effects are obtained when arginin is administered by hypodermic injection except that more nitrogen is excreted in the urine than could have come from the arginin given. 5. The excess of nitrogen does not appear to be caused by the solvent employed, consequently one must assume that arginin stimulates nitrogenous metabolism. 6. The urea-nitrogen quotient (relation of urea-nitrogen to total nitrogen) is increased during arginin administration. 7. In one experiment glycosuria followed the giving of arginin. The cause of the glycosuria is not clear. PROTOCOLS. Exp. 1. Dog, weight 7-6 kgs. Food, minced flesh, 75 grms.; dried bread crumb, 40 grms.; lard, 20 grms. Urine analysis as follows: Total Nitrogen Urea-Nitrogen Phosphates Date in grms. in grms. in grms. Weight. 15. vii * > ,,,, i ,,,, [o X ,,,, ) c ,,,, Arginin carbonate ( grms. N) subcutaneously injected.,,,,,, , ,,,, ' ,,,, Arginin carbonate (0-932 grms. N) mixed with food.,,,,,, ,,,, ,,,, Exp. 2. Dog, weight 6-73 kgs. Food, minced flesh, 75 grms.; bread crumb, 40 grms.; lard, 30 grms. Analysis of urine as follows: Total Nitrogen Urea-Nitrogen Phosphates Date in grms. in grns. in grms. Weight 2. I ' ,,,, ,,,,,, Normal saline 25 c.c. injected subcutaneously. 4.,,,, ,77 5.,,,, 2' ,,,,,, Sodium acetate 25 c.c. of 1 0/0 solution, injected suboutaneouisly. 6.,,, ,,,, * ,,,, 3' ,,,, 2' ,,,, 2'8350 2' ,,,, *

10 146 W. H. THOMPSON. Total Nitrogen Urea-Nitrogen Phosphates Date in grms. in grms. in grms. Weight 12. I ,,,, X ' ,,,, ) ,,,,,, Arginin carbonate (0-84 grm. N) given with food. 15.,,,, * ,,,, Arginin carbonate in neutral solution (0-84 grm. N) injected subcutaneously. 17.,,,, ,,,, ,,,, * ,,,, ,,,, Exp. 3. Dog, weight 7-00 kgs. Food, minced flesh, 75 grms.; bread, 50 grms.; lard, 30 grms. Urine analysis as follows: Total Nitrogen Urea-Nitrogen Phosphates Date in grms. in grms. in gnus. Weight 4. xi ,,,, o ,,,, il i ,,,, ,,,, a ,,,, ) ,,,,,, Arginin carbonate (-7551 grms. N) given with food on this and two following days. 10.,,,, 8S490, ,,,, 86400, 8255 ' ,,,, 87800) 8-89) c ,,,, ,,,, ,,,, ,,,,,, Normal sodium chloride solution injected subcutaneously. 16.,,,, ,,,, ,,,, ,,,,, Arginin carbonate in neutral solution ( grms. N) injected subcutaneously. 19.,,,, : ,,,, ,,,, ,,,,