The Journal of
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`.I|rII:n:.arI 5.-gnaw van Eben:-u-nlfl-|'|v an-a w-Amunr nanny
`
`THE MAXIMUM PRODUCTION OF
`GLUTAMINE BY THE HUMAN BODY
`AS MEASURED BY THE OUTPUT OF
`PHENYLACETYLGLUTAMINE
`
`Carl P. Sherwin, Max Wolf and William Wolf
`J. Biol. Chem. 1919, 37.'113—119.
`
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` LUPIN .
`LUPIN.
`EX. 1016
`EX. 1016
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`THE MAXIMUM PRODUCTION OF G-LUTAMINE BY THE
`HUMAN BODY AS MEASURED BY THE OUTPUT
`OF PIIENYLACETYLGLUTAMINE.
`
`B‘! CARL P. SHERWIN, MAX WOLF, AND WILLIAM WOLF.
`
`(From the Laboratory of Fordham University ll2'c-dire! School, New York.)
`
`{Received for publication, November 12, I918.)
`
`Phenylaeetic acid when fed to animals such as hI:u1'ses (1), dogs,
`rabbits (2), and sheep (3), is excreted in the urine as phenaeeturic
`acid. This eomhillatiml of phenylaectie acid with glyeeeoll, how-
`ever, does not take place in the human body.
`In man the phenyl-
`acetie acid eoiilhilles with glutamine and is excreted in the urine
`as phenylacetylglul.am.ine (4). E. Salkewski and H. S-alkowski
`(2) find after feeding phenylacetie acid to rabbits an increase of
`almost 75 per cent in the total S0; output, but at the same time
`a decrease in the amount of “combined S03.” The increase in
`the amount of inorganic sulfates in the urine they attribute to an
`increased eataholism of protein material necessary for the prepa-
`ration of suflicient amount of glyeocoll to detoxicate the phenyl-
`acetic acid. The decrease in ethereal sulfates they believed to be
`due to the action (if plneruylacetie acid as a disinfecting agent with
`a subsequent decrease in the amount of intestinal putrefat:-lion.
`As glutarnine (5) rather than glutamic ecid seems to be the primary
`ami’n0—-acid constituent of probgin, we thought it iinportarit to de-
`termine the maximum amount of this acid which the human body
`is able to furnish for the detoxication of phenylacetic said. We
`wished also to know whether the ingestion of pheiiyla-2et.ie acid
`would be followed by increased sulfur metabolism in the human
`body as reported by previous investigators (2) in animals. Lastly,
`we wished to determine the effect of the acid in the course of in-
`
`testinal putrefact-ion as measured by the excretion of urinary
`indiean, as well as by the amount of ethereal sulfates excreted in
`the urine.
`
`113
`
`THE JOURNAL 0'!‘ BIOLOGICAL CHEMISTRY. ‘'01.. XXXIVII. SO. 1
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`114
`
`Maximum Production of Glutamine
`
`EXPERIMENTAL.
`
`The subject was a man weighing 60.2 kilos. He was main-
`tained throughout the entire experimental period on a uniform
`diet. The experiment covered 5|. period of % days. On the 5th
`day of this period 2.5 gm. of phenylacetic acid were ingested, on
`the 7th day 5 gm, on the 10th day 7.5 gm., on the 13th day 10
`gm., and on the 21st day 15 gm. The acid in each case was in-
`gested as a solution of the sodium salt. The entire amount of the
`solution was taken at once, covering a period of not more than 3
`to 5 minutes. The urine was collected in 24 hour periods, be«
`ginning at the time of the ingestion of the acid. The urine volume
`was first measured, and sufficient amount taken for the indican
`and sulfate determinations on that day and the day following the
`acid ingestion; the remaining urine volume was measured, exactly
`neutralized with sodium carbonate, and evaporated on the water
`bath to a thick syrup. The determination of the indican was
`made in duplicate upon each urine sample by the method of El-
`linger (6). Each cc. of the Wang solution (7) employed in the
`titration was found upon standardisation to be equivalent to
`0.300 mg. of indigo,‘ or 0.576 mg. of indican. The ethereal sul-
`fates and total sulfates in the urine were determined by the method
`of Folin (8). To determine the quantity of phenylacetylgluta—
`mine, the evaporated urine was made acid with phosphoric acid
`(Congo red as indicator) and extracted in a continuous extracting
`apparatus with absolute ethyl acetate. The amount of ethyl
`acetate used during each extraction was about 300 ee., and the
`time of extraction varied between 1 and 2 hours. The extracting
`was continued until the ethyl acetate was found on evaporation
`to contain no more phenylaeetylglutarnine. To obtain the phen-
`ylacetylglutaminc the ethyl acetate extracts were placed on
`ice for 24 hours. At the end of this time, the white flaky crys-
`tals of the substance appeared on the sides and bottom of the
`flask. These crystals were scraped loose and filtered off,
`the
`mother liquor was evaporated to one-half the volume, and again
`placed in the ice box for 24 hours. This process was repeated
`until all the compound was crystallized out of the solution. The
`
`1 The indigo used in standardizing the Wang solution was furni:-slied by
`Professor P. B. Hawk of Jefferson Medical College.
`
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`191101301101S3T1fi,’5.J0'3qIl"i’!‘\.‘:\.-\\I.'_!:dullLI1D.gp9'pl20[l.Ia!\D(_'[
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`C. P. Sherwin, M. Wolf, and W. Wolf
`
`115
`
`phenylacctylglutamine from the different extractions was united
`and recrystallised from absolute ethyl acetate, dried, and weighed.
`In each extract there occur1'ed about an equal amount of phenyl-
`acetylglutaminc and phcnylacctvlglutamine urea.
`In order to
`split off the urea and convert the urea compound into phenyls.ce-
`tylglutaminc, a water solution of the urea compound was made
`slightly alkaline with barium hydroxide, the solution treated with
`carbon dioxide to remove the excess of barium, filtered, and the
`filtrate evaporated to dryness at a low temperature on the water
`bath. The residue, consisting; of the barium salt of phcnylace
`tylglutamiuc and urea, was extracted several times with hot abso-
`lute alcohol to remove the urea. The insoluble barium salt was
`
`separated from the urea by filtration. The barium salt of the
`phenylaeetylglutauiine was dissolved in a small amount of water,
`made acid with phosphoric acid, the barium sulfate filtered off,
`and the phcnylacetylglutamine extracted from the concentrated
`solution with ethyl acetate.
`
`D 150 U SBION .
`
`The results of the experiment are summed up in Table I.
`On account of the toxicity of the phenylacetic acid, it was im-
`possible for the subject to ingest more than 15 gm. of the acid.
`Even 5 gm. caused thirst, a slight feeling of dizziness, and nausea.
`The 10 gm. dose seemed to produce no exaggei-ation of these
`symptoms, but after ingestion of 15 gm. the subject noticed
`marked signs of poisoning, not unlike those following the inges-
`tion of large quantities of alcohol. The daily urine volume which
`generally measured 800 to 900 cc. rose to at least 1,000 cc, and
`in one instance to 1,300 cc. after the ingestion of more than 5
`gm. of phenylacetic acid. The increase in sulfur metabolism
`noted by Salkowski and Salkowski (2) after feeding the same acid
`to rabbits is not borne out
`in this experiment.“ On the 13th
`day of the experiment after an ingestion of 10 gm. of the acid,
`there was a rise in the total 30.; output from 1.6862 gm. on the
`previous day to 2.5107 gm. This. although a marked rise,
`is
`even lower than the amount excreted the 3rd day before the inges-
`tion of any of the acid.
`
`5 The effect of phenylacetylglutamine production on nitrogen metabo-
`lism is being studied and will be reported later.
`
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`116
`
`Maximum Production of Glutamine
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`

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`C. P. Sherwin, M. Wolf, and W. Wolf
`
`117
`
`On the 10th day, after receiving 7.5 gm. of the acid, there was
`an increase in total S0; of 0.1404 gm. or 5.95 per cent over the
`previous day.
`In all other cases there is even a decrease in the
`total amount of S04 excreted in the urine. There is no marked
`decrease in the amount of ethereal sulfates after the ingestion of
`the acid except on the 21st day of the experiment, when the
`amount fell to less than half the amount of the previous day and
`was approximately one-hall the amount excreted on the succeeding
`day. There was also a relative decrease in ethereal sulfates in
`every case as is shown by the proportion of total S0; to ethereal
`S04.
`
`The influence of the phenylacetic acid on putrefaetion in the
`intestine is best measured by the indican (9) output in the urine,
`as the amount in each case is aficcted by the ingestion of the acid.
`The influence of the phenylaeetic acid on the indiean excretion is
`most marked on the 1st or 2nd clay after the ingestion of the acid.
`After a 5 gm. dose of the acid, the indican entirely disappeared
`for the same day and the day following, while on the 13th day 10
`gm. of the acid caused only a slight decrease in the amount of
`indiean excreted. After the ingestion of 15 gm., there is a very
`slight drop in the 24 hour indican value followed the next day
`by a marked increase. The decided lowering of the indiean con-
`tent of the urine first appeared on the 2nd and 3rd days after
`acid was taken. The ratio of indican S0,; to total ethereal S04
`shows that the production of indican was retarded to a. much
`greater extent
`than the other compoimds constituting the re-
`mainder of the ethereal sulfates.
`
`In every case the amount of glutamine furnished by the body
`for the detoxication of the phenylacetic acid was approximately
`one-half the necessary amount. Regardless of the size of the
`dose of acid, only about 50 per cent of the acid appeared in the
`urine as pheriylacetylglutarnine.
`After a dose of 2.5 gm... 2.02 gm. of the compound, correspond-
`ing to 52.88 per cent of the acid ingested, were isolated from the
`urine. After ingesting 15 gm. of the acid, 14.75 gm. of phenyl-
`acetylglutarnine were recovered from the urine.
`In this case
`50.65 per cent of the acid ingested was recovered. An exception
`was found after a 5 gm. dose, when the amount of acid regained
`amounted to 67.67 per cent with a. collection of 3.3820 gm. of the
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`118
`
`Maximum Production of Glutamine
`
`compound. The amount of gllltalnine furnished in each case
`was proportional to the amount of phenylacetic acid ingested
`1‘i.3}2‘_E].1"dlBSS of the size of the dose. The largest quantity of gluta-
`mine isolated from the urine in coinhination with the phenylacctic
`acid was 7.5225 gut. after a dose of 15 g:11.nftl1e acid.
`It is prob-
`ahlo that more nf the phcn_ylaccty]glutamine would have ap-
`pez1.red in the urine after em.-.lI dose of the H.l'.:il.l, had the acid been
`irigeslml at regular intervals covering 3 period of 10 or 12 hours.
`Thert-. nmy have br_'c1J a rcve1'sil.)le reaction at work thus decreas—-
`ing the aiuount oi the phenylacetits. acid compound in the urine,
`or the I'emaindP.r of the acid yet. Ilnaccolintcd for niay have been
`detoxicated and eliminated through the urine as an entirely dif-
`ferent compound.
`
`|'TnNl’}LIl:'-iIflN.
`
`1. No niarkcd increase in sulfur metabolism followed the inges-
`tion of phenylacetic acid by man, as is shown by the Very Slight
`increase in the total S04 content of the urine. This does not agree
`with the findings of the experiment on rabbits by Salkowski and
`Salkowski.
`
`2. Intestinal putrefaction is decreased after ingesting phenyl-
`acctic acid. The amount of urinary indican was decreased as
`well as the total ethereal sulfates.
`
`3. About 50 per cent of the phcnylacetic acid ingested was de-
`toxicated in each case by uniting with glutamine. The largest
`amount of glutamine excreted was 7.5225 gm. after a dose of 15
`gm. of phenylacetic acid. The amount of phcnylaeetylglutaminc
`isolated from the urine after the 15 gm. dose of the acid was
`14.75 gm.
`
`BIBLIOGRAPHY.
`
`1. Salknwski, E, Ber. chem. G'e.~z., 1884, xvii, 31110: Z. physild. Chem.., 1885,
`ix, 229.
`2. Salkowski, E., and Sallmwski, H.. Ber. chem. Gee, 18?9, xii, 653; Z.
`physiol. Che-m., 1882-33, vii, 161.
`3. Vaailiu, llfitt. Tend-in. Inst. Unis. Brestatt, 1909, iv, 703.
`4. Thierfelder, H., and Sherwin, C. P., Ber. chem.
`(z‘c.-3., 191-1,
`Z. jphysiol. C'imn., 191:1, xniv. 1.
`
`jclvii, 2630;
`
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`191101301101S3'[lfi,€io'aq['m.u.\\,;::d11q|.[1D.gp9'|3l20[l.Ia!\DG
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`C. P. Sherwin, M. ‘Wolf, and W. Wolf
`
`119
`
`5. Sherwin, C. P., Inaugural dissertation, Ti'1bingen, 1915. Fischer, E.,
`and Koenigs, E., Ber. chem. G'es., 1904-, xxxvii. 4585. Osborne, T. B.,
`and GilbI31‘t,R. D., Am. J. Pkys'iol., 19U.'.’r—06,xv, 333. Osborne, T. B.,
`Leavenworth. C. S., and Brautlecht, C. A., Am. J. Physiol., I908r—09,
`xxiii, 184. Ahderhalden, E.. Lchrb. physiol. Chem., Berlin, 1915,
`3rd edition, ii, 1169.
`. Ellinger, JL, Z. physfol. Chem., 1903, xxxviii, 192.
`. Wang, E., Z. physiol. Chem. 1898, xxv. 409.
`. Folin, 0., Am. J’. PIe.ys1'o.E., 1905, xiii, 52.
`. Fnlin, Am. J. Phy.s£oi., 1905, xiii, 99.
`
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