THE METABOLISM OF THE AMIDINE GROUP OF ARGINISE IN THE INTACT RAT

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1 THE METABOLISM OF THE AMIDINE GROUP OF ARGINISE IN THE INTACT RAT BY DEWITT STETTRN, Ja., ASI) BEN BLOOM (Prom the National Institute oj Brthritis and Metabolic Diseases, National Institutes of Health, Public Health SertCe, Bethesda, Maryland) (Received for publication, October, 955) The present study is concerned with the metabolism of arginine in the intact rat. The amidine group of the arginine employed was labeled with two isotopes, N5 and C4. It was hoped that by the device of double labeling information beyond that presently available could be secured about the various fates of this amino acid. The first question about which information was sought Teas the possibility of loss and subsequent replacement of amidine nitrogen without concomitant exchange of amidine carbon. This might result, for example, from reversal of the citrulline + arginine reaction (, ) or from the action of an as yet undiscovered mammalian arginine desimidase. Such processes, if they occurred, would be expected to lower the ratio N5/C4 in the arginine of tissue proteins. The nitrogen of the amidine group of creatine is knolvn to arise from t.he amidine group of arginine (3, 4). It has not been established, however, whether in this transamidination reaction the amidine group migrates with I its skeleton (=N-C=N-) intact. Equality of the ratio N5/C4 in the amidine group of carcass creatine with the corresponding ratio in administered arginine would support the conclusion that C-N bonds within the amidine group were not ruptured incident to this transfer. Studies with arginine-amidine-n l5 (4) have yielded results in excellent agreement with the classical hypothesis of an ornithine-citrulline-arginine cycle. When urea-n5 was fed, it appeared to behave as a metabolic endproduct, little N5 being recovered in products other than urea. We know of no studies in which the fate of the amidine-carbon of arginine has been similarly followed, but several investigators have reported on the administration of urea-c4 (5, 6), and have found that a considerable fraction of this carbon is recoverable in expired COZ. The present experiments with doubly labeled arginine were designed to shed light on this apparent discrepancy.

2 74 AMIDINE GROUP OF ARGININE EXPERIMENTAL Synthesis oj 8~yilline-drnidine-Cl4,-P liyclrochloride-the synthesis of L-srginine monohydrochloride, doubly labeled in the amidine group with Cl- and Y5, followd in general the method of Murtx (7). This method involves the condcneat,ion of the caoppcr complex of ornithinc with O-methylisouronium chloride. Cyanogen bromide-u \vas prepared by addition of 4 mmoles of NaCN in a total volume of 4 ml. of H,O (8) to 40 mmoles of Brz, in a flask immersed in ice. The addition, which was made over a 4 hour period with stirring, consisted first of mmole of NaCY4N ( mc.),l followed by 40 mmoles of non-isotopic NaCN. During addition the temperature was maintained at 0 or below. The crystalline product was then distilled through a short stem into a receiver immersed in ice and fitted with a reflux condenser. All the material which could be distilled on a boiling water bath WLS collected. The crystalline distillate, which was slightly brown in color, was dissolved in 30 ml. of ether and treated with anhydrous CaClz for hour at 0. To the filtered ethereal solution of crude C? NBr was added a chilled solution containing 80 mmoles of W5H, (approximately G atom per cent excess) in 60 ml. of absolute methanol (9). A colorless solution resulted which, upon warming to room temperature, deposited crystals of N5H4Br. On the following day the crystals were removed by filtration, the filtrate was evaporated to dryness in vacua, and the residue was repeatedly extracted with port ions of boiling ether. The combined ether-insoluble material, presumably IV5H4Br, weighed 3.43 gm. (88 per cent of theory). The ether-soluble HZN-C=N was recovered after removal of solvent under XX and dried in vacua over I 05. It lyeighed.30 gm. (3 mmoles). This crude cyanamide was treated with about 5 ml. of cold absolute CHsOH and.5 gm. of anhydrous HCI (0). The mixture was left at room temperature for 5 days in a flask protected by a CaC& tube. The solvent was then removed under Nz, and the residue was dried. This amounted to 3.07 gm. (8 mmoles calculated as 0-methylisouronium chloride). 30 mmoles (6.5 gm.) of L-ornithine dihydrochloride ([or]: 4.35, concentration 4 per cent) (7) were suspended in water and brought into solution by adjustment of the ph to 7.0 with N NaOH. This clear solution (about 0 ml.) was treated with an excess of basic copper carbonate, boiled for 5 minutes, filtered, and cooled. G ml. of N NaOH were then added, and t,he entire solution of copper-ornit,hine complex was transferred onto Obtained from Tracerlab, Inc. Obtained from the Eastman Kodak Company.

3 D. STETTEN, JR., AND B. BLOOM 75 the dry 0-methylisouronium chloride described above. The resulting clear dark blue solution was kept at room temperature for 7 days. The solution was then brought to ph 3.5 with HCI, and the mixture was saturated with HZS, boiled to eliminate excess HZS, and filtered. To the filtrate plus mashings were added 75 mmoles (6. gm.) of flavianic acid dissolved in 65 ml. of hot water (). Immediate crystallization occurred, and after refrigeration for 4 hours the crude arginine flavianate was collected, washed with ice water, and then with ethanol. The crude arginine flavianate (9 gm.) was decomposed by treatment on the steam bath with 45 ml. of concentrated HCl solution for hours. After refrigeration for hours, the regenerated flavianic acid was removed by filtration, and the filtrate and washings were evaporated to a syrup in vacua, dissolved in boiling water, and decolorized with charcoal. Ethanol was cautiously added to the filtrate, the ph adjusted to about 7 by addition of aniline (la), and t he resultant oily precipitate seeded and scratched. After several days of refrigeration the precipitate was collected, washed with cold ethanol, and dissolved in 0 ml. of hot water. After decolorization with charcoal, the solution was treated with ethanol, and, upon seeding, white crystals of n-arginine monohydrochloride were deposited. After refrigeration these were filtered off, washed with ethanol, and dried in vucuo, first over CaC&, then over PZOG. The final yield was 3.48 gm. (6.5 mmoles = 4 per cent of theory based on. 40 mmoles of starting materials). The nitrogen content (Kjeldahl) of the arginine monohydrochloride was 6.6 per cent (calculated 6.8 after correct,ion for 5 atom per cent excess W5). [a]?.6 7. ( concentration.6 per cent as arginine in 6 N HCl). From the accumulated mother liquors of the last stages, an additional.0 gm. of recrystallized arginine nonoflavianat.e were secured. Feeding Experiments-Male rats of the Sherman strain weighing from 80 to 0 gm. were divided into three groups. Experiments -A and -B consisted of three and two rats, respectively. The rats of Experiment -A were maintained in a metabolism cage which permitted daily collection of urine. The rats of Experiment -B were maintained in a desiccator modified to permit daily collection of expired COZ. The urines of this latter group were preserved with diphenylmercuric borate and pooled for the duration of the experimental period. The treatment of the three rats in Experiment was identical with that in Experiment -A. For 3 days prior to experimentation the rat,s were given access to a diet consisting of 8 per cent casein, 5 per cent salt (Osborne-Mendel mixture No. l), 55 per cent corn starch, 7 per cent cellulose, 5 per cent yeast, 8 per cent corn oil, and per cent cod liver oil. To minimize scattering, the diet was made into a paste with water. At the beginning of the experi-

4 76 AMIDINE GROUP OF ARGININE mental period, sufficient isotopic arginine monohydrochloride was added to the diet so that the animals received an average of.5 mmoles of arginine per kilo of rat per day. The feeding of the isotopic diet was continued for 3 days. At the end of this period t,he tissues of rats of Experiments -A and -B were combined. Urine-Ammonia nitrogen was determined and isolated for N assay by aeration after addition of KzC03 (3). nitrogen was measured by a urease method (3). The NL5 and Cl4 contents of urea were obtained by analysis of the dixanthydryl derivative (4). The nitrogen contents (Kjeldahl) of the dixanthydryl urea derivatives ranged from 6.7 to 6.9 per cent (calculated 6.7). Nucleic Acids, Arginine, Aspartate, and Glutamate-Sodium nucleates were isolated from the internal organs (kidney, liver, thymus, testis, and spleen) by extraction with hot 0 per cent NaCl solution as described by Conzelman et al. (5). The product did not contain sufficient isotope to warrant further study. The residue was defatted by extraction with alcohol and ethyl ether and was dried. Arginine monoflavianate was isolated essentially as described by Vickery () and converted to arginine monohydrochloride according to the method of Cox (). The nitrogen content of the isolated arginine monohydrochloride was 6.6 per cent, Experiment, and 6., Experiment (calculated 6.6). The mother liquor from the arginine flavianate isolation was freed of flavianic acid by neutralization with Ba(OH) solution, filtration, and further treatment with charcoal. Glutamic acid hydrochloride and cupric aspartate were isolated as described by Stetten and Schoenheimer (6). The nitrogen content of the glutamic acid hydrochloride was 7.7 per cent (calculated 7.6) and of the cupric aspartate 7.3 (calculated 7.). Creatine-After removal of the organs and skin the carcasses were minced, and creatine was isolated as creatinine picrate according to Foster et al. (7). The creatinine picrate was converted to creatinine*znclz by the method of Benedict (8). The creatinine.znc contained 3.5 per cent nitrogen, Experiment, and 3.0 per cent, Experiment (calculated 3.). Amidine-N5, -Cl-The isotope content of the amidine group of arginine and of creatine was measured on samples of NH, and COz obtained by refluxing these products with Ba(OH) as described by Bloch et al. (9). Results Recovery of Isotope in Tissue Constituents-Comparison of the concentrations of CY and N5 in the arginine fed with that recovered from internal organs (Table I) reveals an approximate loo-fold dilution to have taken place in each of the experiments. The alkaline degradation of arginine tu

5 D. STETTEN, JR., AND B. BLOOM 77 liberate the C and IV of the amidine group gave the theoretical value for N5 on synthetic arginine, but the value for Cl4 Iv-as slightly less than ex- TABLE Isotope Distribution after Feeding Alginine-Cl4 and -W5 Arginine labeled in the amidine group with Cl4 and Nl was included in the diet of three groups of rats, Experiments l-a, I-B, and, at the level of.5 mmoles per kilo of rat per day for 3 days. The rats were sacrificed, and the internal organs and carcasses from Experiments -A and -B were pooled. For further details, see the experimental section. - Arginine, fed In amidinel Arginine, organs In amidinet Arginine, organs In amidinet Glutamate, organ Aspartate ( Creatine, carcasses In amidinet Creatine, carcasses In amidinet, urine, st day ( nd < 3rd lst-3rd da) I< st day < ([ nd I < 3rd T Experin nent No. -A -B - a (a) km per cent ezcess I hrrected N S* 65) peu cent C Cc) c.p.nz. per milliatonr c 4.59 x 0.56 X X lo3.3 x lo x x X lo3.99 x X lo3.66 x x lo4.46 x lo*.03 x 04.9 x x lo4 3.8 X lo4.55 x 04 per cen * Values in columns (b) and (d) are isotope concentrations adjusted to 00 in the amidine group of arginine fed. f The term amidine refers to NH and CO liberated after treatment with boiling Ba(OH) solution. petted. When the dilutions of the two isotopes of organ arginine are compared (last column, Table I), it would appear that the amidine carbon has undergone slightly greater dilution than the amidine nitrogen. As was anticipated from the results of Bloch (4), some lf was recovered in

6 78 AMIDINE GROUP OF ARGININE the dicarboxylic amino acids of the organs, more in glutamic than in aspartic acid. Nitrogen of the amidine group of creatine is known to arise from that of arginine (4). Evidence published to date does not permit a decision as to whether the amidine group,. comprising C atom and N atoms, is transferred intact. In the present experiments (Table I), the carcass creatine isolated contained both isotopic atoms with which the ingested arginine was labeled. The concentrations of isotope in the amidine group of creatine were less than per cent of the corresponding concentrations in the amidine group of ingested arginine. In each case, however, the ratio of the two isotopes in the amidine of creatine (last column, Table I) did not deviate significantly from that in the amidine of the arginine fed. This finding supports the view that, in this transamidinat ion reaction, the amidine group migrates intact. TABLE II Elimination of Amidine Calbon As COZ The expired COZ from the rats of Experiment I-B was collected in 4 hour portions. Consumed as arginine Excreted as COn Day No - activity Specific activity activity 3 I c.pm. c.p.m. $%r milliatom c c.p.m. 6. X lo x X 0 : x x X lo5 Distribution of Isotope in Excretory Products-Of the Cl4 ingested as amidine-labeled arginine, a considerable fraction appeared in respiratory COz. The specific activity of the CO? rose with the passage of time (Table II). In the 3 day period of study, two-thirds of the ingested radioactive carbon appeared as COZ, and, on the 3rd day of feeding, actually more Cl* was exhaled as COz than was ingested on that day. The magnitude of this conversion was unexpected in view of the fact that, in the accepted pathways of mammalian met,abolism of arginine, no direct route exists from amidine carbon to COZ. Of the N5 ingested as amidine-labeled arginine (Column f, Table III), 35 to 45 per cent was recovered in the total urinary nitrogen (last column, Table III) in the 3 day period. In those experiments in which daily urine collections were made, the ammonia of the urine contained 0.4 to 4 per cent as large a quantity of N5 as did the urea. The N5 concentrations in total urinary N as well as in urea and ammonia rose on successive days. It is of interest to compare the concentrations of Cl4 and of IV5 in urinary urea with those of the arginine fed (Table I). In contrast to the behavior

7 I). S ltlw:~, JR., ASD B. BLOOM 79 of W5, the Cl concentration in urea was irregular, wit,h a tendency to decline which was most marked on the 3rd day of each experiment,. The reason for this decline, in the face of a rising Xl5 concent,ration in urea and a rising specific activity in CO (Table II), is not apparent. This behavior is reflected in the values in the last column of Table I. Here TABLE Partition of Nitrogen and Isotope Distribution in Urinary Products Experiments I-A and were conducted on three rat,s each; Experiment -B was conducted on two rats. III Consumed Urine -A -B nay so Arginine (cl?la?nole sitrogen NH, NH3 XH, NH, NH3 NH, NH, s aom per cent eaxe.ss iy,; (RI x C/L) it is seen that the ratio of isotopes (X5/C? ). in urinary urea, close to unity initially, rises to a value of 3 to 4 by the 3rd day. From this it is clear that, of the constituent atoms of urinary urea, a larger fraction of t.he N t>han of the C is derived from the amidine group of ingested arginine. )scuss0s Three findings which have become apparent in the description of the results are presumably related. These are () that a major fraction of

8 730 AMIDINE GROUP OF ARGININE the amidine carbon of arginine fed to rats appears as expired C0, () that the ratio P5/C?4 in urinary urea rises significantly above the corresponding ratio in the amidine group of ingested isotopically labeled arginine, and (3) that, in the arginine isolated from internal organs, this same ratio appears to be slightly higher than in the material ingested. Whereas no mammalian enzymes are known to account for the direct conversion of amidine carbon of arginine to CO*, there is ample evidence (9) that ureases from bacteria in the gastrointestinal tract may degrade urea, which in turn arises from the action of arginase upon arginine. Of the products of gastrointestinal urea breakdown, the ammonia would be expected to enter the portal circulation, to be returned to the liver for resynthesis into arginine, ultimately appearing in large part as urea. The carbon dioxide, on the other hand, even assuming efficient intestinal absorption, would be mixed with a large reservoir of CO prior to reentry into the urea cycle, and its isotope would be greatly diluted. It is concluded that the metabolic inertia of urea in the intact animal, based on studies with Xl5 alone, is apparent rather than real and reflects the efficiency with which ammonia arising in the gastrointestinal tract is resynthesized into urea in the liver. SUMMARY L-hrginine-amidine-IV5-CY4 has been fed t o rats for 3 days. The ratio, Pp.34, in the amidine group of arginine isolated from organ proteins was somewhat higher than that in the material fed. The corresponding value in the amidine group of t)he carcass creatine was essentially equal to that in the arginine-fed group. The ratio PF5/C4 in the urinary urea of the st day was also approximately equal to that in the amidine of dietary arginine, but rose to values three to four times as high by the 3rd day. Over the 3 day period, two-thirds of the Cl4 ingested was exhaled as C40. These results indicate t)he absence of any process wherein amidine nitrogen of arginine is lost without coincident loss of amidine carbon. The transamidination from arginine ult imately to yield creatine proceeds without rupture of C-X bonds in the amidine group. In agreement with the findings of others, it is concluded that urea, arising from the amidine group of arginine, is subject to extensive catabolism in the intact animal, presumably in the gastrointestinal tract,. Much of the ammonia arising in this process is resynthesized into arginine, whence it reenters the urea cycle, whereas most of the CO is lost in the exhaled air. BIBLIOGRAPHY. Katner, S., in hlci~lro)-, W. II., and Glass, I<., Amino acid metabolism, Baltimore, 3 (955).

9 D. STETTEN, JR., AND B. BLOOM 73. Krebs, H. A., in Sumner, J. B., and Myrbllck, K., The enzymes, New York,, 866 (95). 3. Borsook, H., and Dubnoff, J. W., Science, 9,55 (940). 4. Bloch, K., J. Biol. Chem., 66,469 (946). 5. Dintzis, R. Z., and Hastings, A. B., Proc. Nut. Acad. SC., 39, 57 (953). 6. Chao, F., and Tarver, H., Proc. Sot. Exp. Biol. and Med., 84,406 (953). 7. Kurtz, A. C., J. Biol. Chem., 80, 53 (949). 8. Hartman, W. W., and Dreger, E. E., Org. Syntheses, ~0., 50 (943). 9. Bloch, H., Schoenheimer, R., and Rittenberg, D., J. Biol. Chem., 38,55 (94). 0. Stieglitz, J., Ber. them. Ges., 33, 57 (900).. Vickery, H. B., J. Biol. Chem., 3, 35 (940).. Cox, G. J., J. Biol. Chem., 78, 475 (98). 3. Hawk, P. B., Oser, B. L., and Summerson, W. H., Practical physiological chemistry, New York, 3th edition, 884,889 (954). 4. Fosse, R., Compt. rend. Acad., 67, 948 (93). 5. Conzelman, G. M., Jr., Mandel, H. G., and Smith, P. K., J. Biol. Chem., 0, 39 (953). 6. Stetten, M. R., and Schoenheimer, R., J. BioZ. Chem., 63, 3 (944). 7. Foster, G. L., Schoenheimer, R., and Rittenberg, D., J. BioZ. Chem., 7, 39 (939). 8. Benedict, S. R., J. BioZ. Chem., 8, 83 (94). 9. Kornberg, H. L., and Davies, R. E., Physiol. Rev., 36,69 (955).

10 THE METABOLISM OF THE AMIDINE GROUP OF ARGININE IN THE INTACT RAT DeWitt Stetten, Jr. and Ben Bloom J. Biol. Chem. 956, 0: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at tml#ref-list-