618
`
`GEORGE J. SHIPLE AND CARL P. snsswm
`
`lCoN'rsna'ur1oN sso.-.1 TI-IE Cusmcnn Rsssascu Lanosaronv or Formosa: Usrvsssrrrl
`
`I.
`SYNTHESIS OF AMINO ACIDS IN ANIMAL ORGANISMS.
`SYNTHESIS OF GLYCOCOLL AND GLUTAMINE IN THE HUMAN
`ORGANISM
`BY GEORGE J . SHIPLE AND CARL P. SI-IERWIN
`Received October 31. 1921
`
`The fact that the human body is able to furnish glycocoll for the conju-
`gation with benzoic acid has long been known and has been studied by
`many investigators.
`Lewinskil observed neither benzoic acid nor benzoyl glycurouates in the urine oi
`men to whom 12 to 20 g. of benzoic acid had been fed in small doses during the course
`of 12 hours. After feeding doses of 25 to 40 g., small amounts of benzoic acid (about
`25% of the intake) were excreted as the free acid along with certain dextro-rotatory
`substances of a reducing nature indicating the presence of glycuronates. One may
`conclude from this that the average human adult is able to convert quantitatively
`about 20 g. of benzoic acid into hippuric acid. Brugschf-‘ and Tsuschija.' on the con-
`trary, were able to recover only about 25 to 50% of the ingested benzoic acid ashippuric
`acid. Dakin‘ found in his work with benzoic acid that doses of 5 to 10 g. of the acid
`ingested per day were almost entirely converted into hippuric acid before its elimination
`in the urine. The human body like that of the lower animals is therefore able to syn-
`thesize glycocoll within reasonable limits under conditions where no glycocoll occurs
`in the protein of the diet or even in a protein-free diet. Wiechowski“ maintained that
`the glycocoll content of the tissues is insufficient to account for the quantity of glycocoll
`thus eliminated and that glycocoll can be synthesized at the expense of urea formed
`under normal conditions. Ringer‘ observed in his work an increase in the nitrogen
`elimination after feeding benzoic acid to animals and suggested that glycocoll resulted
`from the "extra destroyed protein” during this period. Epstein and Boo]-:rnar1,7 ex-
`perimenting on rabbits, concluded that benzoic acid as a toxic substance acts in a sc-
`lective way causing the elimination of excessive amounts of nitrogen which is mostly
`excreted as hippnric acid nitrogen. McCollum and Hoagland” brought a pig of 46 .7
`kg. body weight into a condition of minimal nitrogen metabolism by feeding a diet of
`starch containing 75 calories for each kilogram of body weight. The diet was continued
`and increasing amounts of benzoic acid were fed. The results of their experiment
`Sl'l0‘W that when protein metabolism is reduced to a minimal level by carbohydrate
`ingestion the addition of benzoic acid does not affect the creatinine output, affects but
`little the total nitrogen, but may reduce the total elimination of urea nitrogen from 56 ‘$13;
`of the total nitrogen output to 19% of the total. This difierence of 37% of the total
`nitrogen which is ordinarily converted into urea is under these eircum stances eiiminatcd
`as glycocoll.
`Lewis9 kept a man on a low protein diet containing no glycocoll for a period of :5
`
`1 Lewinski, Arch. exp. Path. Pkarm.. 58, 397 (1908).
`3 Brugsch, Z. exptl. Pot-la. T.ixeraf>.. 5, 731 (1909).
`3Tsuschija,
`fb£d., 5, 737 (1909).
`4 Dakin, J. Biol. Chem.. 3. 103 (I909).
`5 ‘Wiechowski, Be-itr. chem. Pl.-.ysiol., 7, 204 (I906).
`e Ringer. J. Biol. Chem, 10, 327 (1911).
`7 Epstein and Bookman, ibid., 10, 353 (1911).
`“i\-IcCollurn and Hoagland.
`r'bt'a',., 16, 299 (1913-1914).
`9 Lewis.
`ib:'cl.'.. 1s, 225 (1914).
`
`PAR PHARMACEUTICAL, INC.
`EX. 1017
`PAR PHARMACEUTICAL, INC.
`EX. 1017
`
`

`
`SYNTHESIS OF AMINO ACIDS IN ANIMAL DRGANISMS
`
`619
`
`days. On the second day the subject ingested 8. 4? g. of benzoic acid and the urine was
`collected at 2-hour intervals. Urea nitrogen and ammonia nitrogen were determined
`as one. During the first 2-hour period the amount of this nitrogen dropped from 80%
`to 07% of the total nitrogen; during the second 2-hour period. to 61.8%, and during
`the third to 64 .9%, after which it returned to normal. This shows that the ingestion
`of 8 .47 g. of benzoic acid may increase slightly the output of total nitrogen in the urine
`but at the same time cause a decided decrease in the amount of urea nitrogen plus
`ammonia nitrogen eliminated.
`Thierfelder and Sherwin‘-° have shown that glutamine is furnished by the human
`body for the detoxicatiorl of phenyl acetic acid and it was later found by Sherwin, M.
`Wolf and W. Wolf,“ that 7 .5 g. of glutamine was synthesized by the body on glutamine-
`(glutaniic acid) free diet for the detoxication of ingested phenylacetic acid without me-
`terially increasing protein metabolism as shown by the total nitrogen and sulfur elimi-
`nation.
`
`In previous work a human subject had been reduced to a low protein
`diet“ for a period of only 3 days and benzoic acid fed on only lday.
`It
`seemed to us important that the subject be reduced to an endogenous
`level of nitrogen metabolism, if possible, and the experiment be extended
`over a longer period. We also decided to feed first benzoic acid, then
`phenylacetic acid and lastly a mixture of the two in order to answer if
`possible the following questions:
`(1) What is the maximum amount of
`urea nitrogen which the human body will "side track” for the synthesis
`of glycocoll, and what influence if any has this on the other nitrogen con-
`stituents of the urine?
`(2) Can glutamine be synthesized at the expense
`of the urea nitrogen alone or does the amino and amide nitrogen necessary
`for this compound come from different sources?
`(3) Can glycocoll and glu-
`tamine be formed simultaneously in the human body, and if so which
`one is more readily formed when a limited supply of urea nitrogen is avail-
`able?
`
`The subject, a healthy man of 80 kg. body weight, was maintained on
`:1 diet of cream, bananas, and starch or lactose for a period of 7 days.
`
`TABLE I
`
`Isoesrsn Foon PER DAY, AND rrs Emsxor VALUE
`
`Period Starch
`Days elapsed G,
`1
`I80
`2
`St’)
`3
`100
`4
`100
`5
`40
`6
`40
`7
`40
`
`Lactose Bananas Cream
`C.
`G.
`G.
`0
`50
`435
`120
`325
`100
`120
`380
`30
`110
`420
`10
`200
`5-15
`.3
`200
`335
`0
`150
`450
`0
`
`Total
`energy
`Calories
`2475
`1186
`1314
`1278
`1581
`1276
`1197
`
`Nitrogen in-
`gested in
`food
`G.
`0.030
`0 .520
`0.610
`0.670
`0 .870
`0 .535
`0 .720
`
`'9 Thierfelder and Sherwin, Ber., 47, 2630 (1914); Z. pkg.-séol. Chem” 94, 1 (1915).
`11 Carl P. Sherwin, M. W'oli and W. Wolf. J. Biol. Ch.-am, 37, 113 0918}.
`
`

`
`620
`
`GEORGE _]. SHIPLE .‘\.\-'1) CARL P. SHERWIN
`
`
`
`
`
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`
`

`
`‘SYNTHESIS 01:‘ amino acms IN ANIMAL oaoamsns
`
`621
`
`The preceding table shows the weight of each substance eaten per day
`as well as its calorific value.
`
`This diet which is practically nitrogen free insofar as utilizable nitrogen
`is concerned" is also of sufficient calorific value for the needs of a man
`
`undergoing only a small amount of physical exertion. Agar-agar was
`ingested in sufiicient amounts" to cause the regular evacuation of the in-
`testine and thus avoid the absorption of nitrogenous end products of pu-
`trefaction.
`
`In Table II, the days of acid feeding have been divided into sub-periods
`of varying length in order to study the amount of different substances
`excreted immediately after the ingestion of a given amount of acid. The
`acid ingested during a. certain sub-period of the day was always taken
`at the beginning of that sub-period, and the entire output of urine collected
`during each sub-period was treated as a single unit. Also for each one
`of the last 4 days a sum total is given of all the sub-periods that the entire
`24-hour" period may be compared to one of the first 3 days of the control
`period.
`A brief survey of Table II brings out clearly several interesting facts.
`Neither benzoic acid nor phenylacetic acid when fed in moderate doses
`(3 to 10 g.) to man acts as a stimulator of endogenous metabolism when
`the subject has been reduced practically to a state of endogenous protein
`catabolism. This is shown by the fact that the amount of total nitrogen
`excreted is not increased during the period of acid ingestion.
`It is also
`seen that neither glycocoll nor glutarnine is built at the expense of “extra
`destroyed protein” but rather at the expense of one or more of the ni-
`trogenous constituents of the urine, and of these only urea is afiected to
`a marked degree.
`Urea, during the control period, maintained an average of 74 to 77%
`of the total nitrogen, while during the feeding period it dropped to 60%,
`57%, then to 48% and finally to 28% of the total nitrogen of the entire
`24-hour period.
`If taken by sub-periods even more striking evidence is
`found of the inroads made on urea nitrogen for the purpose of glycocoll
`or glutamine synthesis. After a dose of 3 g. of benzoic acid on the fourth
`day, followed at the end oi 6 hours by a. second dose of 3 g. the urea ni-
`trogen dropped to 48% and 52% respectively of the total nitrogen. On
`the fifth day 2 doses each of 3. 3 g. of phenylacetic acid caused a further
`drop of the urea nitrogen to 39% and 35% respectively of the total ni-
`trogen.
`It is interesting here to note that the effect of the benzoic acid
`on the urea nitrogen was immediate and largely confined to the sub-
`period at the beginning of which it was fed, while the greatest efiect of
`the phenylacetic acid was seen in a later sub-period. One may infer from
`this that either the benzoic acid is more quickly absorbed than the phenyl-
`“ Lusk, "Science of Nutrition,” W. B. Saunrler and Co., 1919, p. 355.
`
`

`
`622
`
`oaonos: _]'. SHIPLE AND CARL P. snakwm
`
`acetic acid, or that glycocoll is more easily synthesized by the body than
`is glutamine. This latter inference one may explain on the ground that
`glutarnine not only demands 2 atoms of nitrogen for its synthesis but be-
`sides an amine requires also an amide group.
`
`During the seventh day of the experiment the greatest reduction is found
`in the urea nitrogen. At the beginning of the fi1’Sl'. 4-hour sub-period
`4 g. of phenylacetic acid was ingested. This reduced the urea nitrogen
`to 44% of the total nitrogen, but caused a still greater fall in urea nitrogen
`(24% of total nitrogen) during the subsequent 4-hour sub-period. At
`this time a second dose of 6 g. of phenylacetic acid was ingested by the
`subject, but the percentage of urea nitrogen rose to 29%, only to fall 4
`hours later to the very low value of 12% of the total nitrogen. This value
`was considerably lower than had been previously obtained. McCo1lurn3
`found that the urea nitrogen fell to only about 20% of the total nitrogen
`after the feeding of large doses of benzoic acid to a pig, and that larger
`doses of the acid caused only an increase in protein metabolism and a sub-
`sequent increase in total nitrogen excreted.
`In this case, however, the
`entire 24 hours was considered as a unit. On the seventh day of our ex-
`periment it will be seen that the urea nitrogen for the entire 24 hours
`formed 28% of the total nitrogen.
`The ammonia nitrogen was little, if at all, affected by the feeding of
`either of these organic acids.
`It has been previously shown by McCollum
`that an organic acid is detoxicated at the expense of the urea nitrogen,
`and an inorganic acid (hydrochloric acid} at the expense of the ammonia
`nitrogen.
`It seemed probable that the amino nitrogen for glutarnine synthesis
`would be taken from the urea fraction, but just possible that the am-
`monia nitrogen might be required to build the amide portion of the mole-
`cule. That this is not the case is shown by the fact that there is no de-
`crease in ammonia nitrogen after the ingestion of 10 g. of phenylacetic
`acid on the last day of the experiment.
`Creatinine was unaffected by the acid ingestion, for the creatinine
`value remained particularly constant during the last days of the experi-
`ment.
`
`Lewinski‘ found that 10 to 20 g. of benzoic acid ingested by a man and
`quantitatively converted into hippuric acid may remove as much as 35%
`of the total nitrogen in the form of glycocoll (hippuric acid) nitrogen.
`Ringer‘ obtained much the same results with goats. Wiechows]-xi,‘ em-
`ploying guinea pigs, found that glycocoll nitrogen might run as high as
`64% after the feeding of benzoic acid. After feeding the 6 g. of benzoic
`acid on the fourth day of our experiment 10% of the total nitrogen was
`found in the form of hippuric acid nitrogen. On the fifth day 19% of
`the total nitrogen appeared in the form of glutamine nitrogen. On the
`
`

`
`SYNTHESIS OF AMINO ACIDS IN ANIMAL ORGANISMS
`
`F123
`
`sixth day after feeding 3. 3 g. of phenylacetic together with 3 g. of benzoic
`acid. 6% of the total nitrogen appeared as glycocoll nitrogen and 119'}.
`as glutamine nitrogen. On the seventh day 35% of the total nitrogen
`was excreted in the form of glutamine nitrogen, which is comparable. to
`figures found by some of the other investigators. Only in certain sub-
`periods of certain days does the excretion of glycocoll and glutamine ni-
`trogen reach the value quoted by Wiechowski.
`In the first sub-period
`(3 hours) of the Sixth day after the ingestion of 3.3 g. of phenylacetic
`acid and 3 g. of benzoic acid, 29% of the total nitrogen was found in the
`form of glycocoll nitrogen and 29% in the form of glutamine nitrogen,
`or a total of 58%. Again in the fourth sub-period (4 hours) of the seventh
`day after the ingestion of 10 g. of phenylacetic acid 52% of the entire
`nitrogen output of the sub-period appeared in the urine as glutamine
`nitrogen.
`Moderate doses of benzoic acid are probably excreted quantitatively
`in the urine as hippuric acid. We found 89% to 93% so excreted within
`6 hours after a 3g. dose. Phenylacetic acid seems to require about twice
`as much time as benzoic acid for its excretion (in the form of phenyl-
`acetyl glutamine).
`Its detoxication, moreover is apparently not so com-
`plete, for though after a dose of 10 g. we were able to recover 95% of it
`from the 24-hour sample of urine, still in other cases after small doses
`we recovered only about 78% of the amount fed.
`
`Apparently the body builds both glutamine and glycocoll simultaneously
`as easily and efficiently as either of the compounds alone. After the in-
`gestion of 3 g. of benzoic acid 92% of it was converted into hippuric acid
`within 6 hours. After the ingestion of 3.3 g. of phenylacetic acid on
`another day 44% of it was converted into phenylacetyl glutamine within
`6 hours. When 3 g. of benzoic acid and 3.3 g. of phenylacetic acid were
`ingested simultaneously 89% of the benzoic acid and 46% of the phenyl-
`acetic acid were changed into their respective detoxication products
`within a period of 6 hours.
`
`Summary
`
`It has been shown that man will synthesize glycocoll at the expense
`of urea as do the lower animals. The Synthesis of another amino acid
`(glutamine) at the expense of urea nitrogen has also been demonstrated
`in the case of a man. The two amino acids may be built simultaneously
`as readily as either compound alone.
`During the period in which these amino acids were being synthesized
`in the organism, urea nitrogen dropped from about 75% of the total
`nitrogen to 28%, and during a sub-period of a certain day, to the extremely
`low value of 12% of the total nitrogen.
`After feeding a moderate dose of henzoic acid (3.3 g.), glycocoli for its
`
`

`
`624
`
`E. P. KOHLER AND L. I. SMITH
`
`detoxication is built within 6 hours, while for the detoxication of a corre-
`
`sponding dose of phenylacetic acid a somewhat longer period of time is
`required for the synthesis of glutamine.
`NEW Yonx, N. Y.
`
`[CONTRIBUTION FROM THE CHEMICAL LABORATORY OF HARVARD UNIVERSITY]
`
`THE REACTION BETWEEN ALKALIES AND CERTAIN N!TRO-
`CYCLOPROPANE DERIVATIVES
`
`BY E. P. KOHLER AND L. 1. SMITH
`Received November 25. 1921
`
`The action of alkalies on all known cyclopropane derivatives is peculiar.
`A typical reaction is that between pheI1yl-benzoylmitrocyclopropane
`and potassium hydroxide which gives potassium nitrite and an open chained
`B-diketone}
`CgH;CH—CHCOC.H,
`\/
`CHN02 + KOH=C;,l-I.CHzCOCI-IaCOCaHn + KNOS + H10.
`
`Since the reaction involves the elimination of the nitro group, it is con-
`ceivable that the first step in the process might be the replacement of this
`group by hydroxyl. This would give as the primary product a cyclopro-
`panol derivative; but it is probable that cyclopropanols are unstable and
`like the corresponding ethylenic compounds immediately undergo re-
`arrangement, for when zinc removes bromine from dibromo-isopropyl
`alcohol the product is not the cyclopropanol which would be expected
`but allyl alcohol,’ and when cyclopropyl amine is treated with nitrous
`acid the product is likewise the unsaturated and not the cyclic alcohol?
`
`CI-IgBrCHOHCHgBr—2Br—r [ CH}-—CH, "I :)- CI-I-_»=CH—CH20H
`
`I
`\/
`CHOH .1
`
`CH; —CI-INH3
`
`\/
`
`CH3+HNO,—J- FCHa—'CHOH]
`
`\/
`CH:
`
`L.
`
`-—J" CHg=CH—CHgOH.
`
`A cyclopropanol obtained by replacing the nitro group with hydroxyl
`would therefore probably be unstable, but it would not be expected to
`rearrange into anything related to_the dike-tone that is obtained:
`CgH5CH—CHCOC‘;H5 :3‘ CaHaCH—CHCOCgH.----P CsH5CH=—C(C0CoHh)CH2OH,
`\/
`\/
`cnnog
`CHOH —>-
`or c.H,cocH=c(c.H.)cH.on.
`
`‘Tins JOURNAL, 41, 1383 (1919).
`‘J’. prakt. C}wm., 4-6, 158 (1892).
`'Zemr., 76,
`[1] 1709 (1905).