ON THE METABOLISM OF HYDANTOINS AND HYDANTOIC ACIDS.

BY OLIVER HENRY GAEBLER AND ANNA K. KELTCH.

(From the Biochemical Laboratory, State University of Iowa, Iowa City.)

(Received for publication, September 11, 1926.)

Interest in the biochemical behavior of hydantoins originated in studies in which the corresponding open chain compounds- the uramido acids-were obtained from urine. Before methyl hydantoic acid1 had been synthesized, its presence in the urine as a product of sarcosine metabolism was reported by Schultzen (1). This &ding was of simultaneous interest in connection with the formation of uramido acids, and with the course of urea and uric acid formation. The latter two excretory products, according to Schultzen’s observations, disappeared on administration of sarco- sine. The comparative behavior of urea, carbamic acid, and po- tassium cyanate, towards sarcosine in vitro was studied soon afterwards (2, 3). The disappearance of urea as an excretory product was traced to the interference of unchanged sarcosine with the method used for determining urea (4). Only slight evi- dence finally remained for the formation of methyl hydantoic acid from sarcosine in the body (Schiffer (5)). Lippich (6) and Dakin (7) point out that another uramido acid or its anhydride thought to be synthesized in the body was probably formed in the procedures used to isolate it.

The hydantoins have, however, been studied in a different connection by Lewis (8). The hydantoin nucleus is present in allantoin, while creatinine and other compounds of biological interest are closely related to it in structure. In a series of papers, Lewis came to the conclusion that the hydantoin nucleus is not attacked in the body.

The experiments with methyl hydantoin here reported were

1 “Methyl hydantoic acid” designates the p-methyl acid, and “methyl hydantoin” its anhydride, throughout this paper.

763

by guest on June 23, 2020http://w

ww

.jbc.org/D

ownloaded from

Metabolism of Hydantoins

undertaken as the result of a suggestion made to one of us some years ago by Professor V. J. Harding, of the University of Toronto, that the possibility of conversion of methyl hydantoin to creatinine by addition of ammonia in the kidney be put to experimental test, Further stimulus was afforded by the observation that methyl hydantoin bore some similarity to blood “creatinine” in the fail- ure of kaolin adsorption, comparative ease of the color reaction with picric acid made alkaline with sodium-carbonate, and in the prolonged character of the reaction. Quantitative considerations in the last connection soon disposed of the possibility that any large proportion of blood “creatinine” could be methyl hydantoin, and with the discovery of extensive destruction and oxidation of the latter compound in the body, our interest shifted to the course of this metabolism.

EXPERIMENTAL.

1. Procedure and Methods.

Two dogs were used as experimental animals. They were kept on the experimental diet for some days before the period of obser- vation began. Urines were collected in 24 hour periods, toluene being used as preservative. All periods were terminated by cathe- terizing and washing the bladder with sterile saline. Total nitro- gen was determined by the Kjeldahl-Gunning procedure, ammonia by permutit (9), urea by the method of Van Slyke and Cullen (lo), creatinine and creatine by the method of Folin (ll), allan- toin in one experiment by Wiechowski’s (12) method, oxalic acid by the method of Dakin (13), and organic acids in several instances by the method of Van Slyke and Palmer (14). In view of the characteristic reaction of methyl hydantoin with alkaline picrate (15, 16), a simple procedure was adopted for approximate colori- metric determination of methyl hydantoin and methyl hydantoic acid in the urines. A control urine will show nearly the same creati- nine value by the Folin method, whether the color development is continued for 10 minutes or 2 hours; a slight fading was the rule in the controls. The same urine with methyl hydantoin added gives greatly increasing values. For example, a control urine in which creatinine was determined by allowing the color to develop before dilution, in separate determinations, for 10 minutes, 1 hour, and 2 hours, gave values for the day of 243, 244, and 239

by guest on June 23, 2020http://w

ww

.jbc.org/D

ownloaded from

0. H. Gaebler and A. K. Keltch 765

mg. The same urine with the equivalent of 1.5 gm. of methyl hydantoin per 24 hour sample added, gave values of 322,737, and 1040 mg. Methyl hydantoin excreted unchanged was therefore taken to be the amount required to be added to control urines of preceding and following days to duplicate the color curve of the urine of the experimental day. If methyl hydantoic acid is pres- ent the “hydrolyzed” urine will show the phenomenon of greatly increasing values, when treated in the above manner, and the amount of methyl hydantoin added to hydrolyzed urine of con- trol days to duplicate the color curve of the hydrolyzed urine of experimental days, was taken as “total methyl hydantoin” or methyl hydantoin plus methyl hydantoic acid excreted unchanged. Hydrolyses were carried out with picric acid (11) in all cases. The values at 10 minutes-which appear in the tables as apparent rises in creatinine and creatine-are worthless in determining the amount of methyl hydantoin and methyl hydantoic acid excreted unchanged, because the additional color is a small part of the total, and because fluctuations in creatine excretion interfere. But the large increases above indicated during the 1st and 2nd hour, make the determination easy. Fortunately, also, where methyl hydantoic acid was encountered as a product of methyl hydantoin, the amount was almost equal to that of methyl hy- dantoin excreted unchanged, -thus making the difference between color curves of unhydrolyzed and hydrolyzed urines of experimental days very large.

Methyl hydantoin and methyl hydantoic acid were prepared from creatinine by the method described in the preceding paper (16). Hydantoic acid was prepared from glycocoll and urea by boiling with baryta as in the method of Baumann and Hoppe- Seyler (2), excepting that separation of the barium salt by use of alcohol was omitted. Barium was removed from the products of hydrolysis by means of sulfuric acid in hot solution and the hy- dantoic acid allowed to crystallize from the filtrate. Hydantoin was prepared from hydantoic acid by evaporation with hydro- chloric acid. All preparations of the four compoundsgaveentirely satisfactory nitrogen values, and the acids gave correct titration values.

by guest on June 23, 2020http://w

ww

.jbc.org/D

ownloaded from

766 Metabolisrp of Hydantoins

2. Metabolism Experiments.

Experiment 1 (Table I).-White, short haired female. Weight 10 kilos. The animal was on a diet’ of 200 gm. of bread and 450 cc. of milk daily, which was consumed quantitatively. On September 27, 1.5 gm. of methyl hydantoin were given subcutaneously in three equal doses, at noon, 5.06 p.m., and 10.30 p.m. The rise in total nitrogen excretion accounts for the nitrogen of the compound given. The compound did not affect ammonia, urea, uric acid, creatinine, creatine, or allantoin excretion. The color curve, with alkaline picrate, of the urine of the experimental day could be duplicated by adding the equivalent of only 15 per cent of the

Date.

1986

Sept. 26 “ 27

“ 28 “ 29

.-

2 2 g

- wn. 4. I( 4.5;

4.M 4.0:

gn.

0.2: 0.24

0.25 0.21

-

i L

!

- 3

d z I 6 -

gm. 3.2! 3.4:

3. I! 3.4f

T7.

294 312

304 312

-

TABLE I.

ctog 1. -

--~

ml. gn. gm.

133 0.1700.033 95 0.1560.033 1.5 gm. methyl hydan-

toin in three doses subcutaneously.

123 0.1700.029 52 0.1690.036

amount of methyl hydantoin given to an aliquot of the control urines.

Experiment 2 (Table II).-The same animal was used as in Experiment 1. The diet was changed to 125 gm. of bread and 300 cc. of milk. It was consumed quantitatively. On November 16, 4 gm. of methyl hydantoin were administered subcutane- ously in three equal doses. The rise of total nitrogen excretion accounted for all of the nitrogen of the compound. Minor rises in ammonia and urea excretion were noted. The rises in creati- nine and creatine excretion are only apparent, and are due tomethyl hydantoin and methyl hydantoic acid respectively. The amount of methyl hydantoin excreted unchanged was estimated colori- metrically at 625 mg., or 15.6 per cent of the amount given. The

by guest on June 23, 2020http://w

ww

.jbc.org/D

ownloaded from

0. H. Gaebler and A. K. Keltch 767

amount excreted as such and as methyl hydantoic acid was 1175 mg., or 29.4 per cent of that given. An aliquot of the urine was evaporated with hydrochloric acid and the residue extracted with chloroform (16). Evaporation of the chloroform left a crystal- line residue, which was recrystallized from absolute alcohol. The crystals melted at 151-155’, and weight for weight gave the same

TABLE II.

Dog 1.

Date.

19.25 gm. gm. gm. gm. ml.

Nov. 14 1.88 1.77 223 “ 15 2.46 0.22 1.88 1.70 220 “ 16 3.48 0.30 2.17 1.93 271

&‘ 17 2.47 0.20 1.86 1.76 231

Date.

1986

Dec. 1 “ 2 u 3

9m.

2.86 2.93 3.70

--- g*. gm. wag.

0.17 2.03 220 0.12 2.16 228 0.22 2.08 288

2.72 0.17 1.99 224 -

nag. cc. 0.1 N 11 164 8 172

44 260 4.0 gm. methyl hydan- toin in three doses subcutaneously.

0 154

TABLE III.

Dog 1.

w. 19 38 69

26

Remarks.

w. 5.6 7.8

355.0 4.0 gm. methyl hydan- toin in three doses subcutaneously.

47.2

intensity of color with alkaline picrate at 10 minutes, 3 hour, and 1 hour as known methyl hydantoin. The amount of recrystal- lized product, calculated to 24 hour volume, was 375 mg. Nearly half of the compound is lost in the amount of alcohol required to remove the pigment and syrupy material, and in any case the amount is much greater than we could isolate from a control

by guest on June 23, 2020http://w

ww

.jbc.org/D

ownloaded from

768 Metabolism of Hydantoins

urine after adding only 625 mg. The presence of methyl hydan- toic acid above indicated was therefore regarded proven.

Experiment 3 (Table III) .-The animal and diet were the same as in the previous experiments. 4 gm. of methyl hydantoin were given subcutaneously on December 3. Less than usual was destroyed, the amount excreted as methyl hydantoin being 30 per cent, and that excreted as methyl hydantoic acid 25 per cent of the amount given. The immense amount of oxalic acid found by the method of Dakin (13) suggested the possibility that methyl hydantoin might be oxidized to methyl parabanic acid, which would be decomposed during the alkaline precipitation of the urine in the oxalic acid determination.

Experiment ,J (Table IV).-The same animal was used. The diet was unchanged except for daily addition of 2 cc. of concen-

TABLE IV. Dog 1.

19s Feb. 19

‘( 20

“ 21

Free Total Total N. oxalic oxalic

acid. acid. --_

om. w7. 4.28 4.80 144.0

3.80 I 45.0

m!?. 2.7

306.0

75.0

Remarks.

4.0 gm. methyl hydantoin in three doses subcutaneously.

trated HCl, which kept the urine just acid. In addition to the other procedures, the following was used in an attempt to decide whether the oxalic acid found was free or combined. 100 cc. of urine were acidified with 5 cc. of concentrated HCI, evaporated to 25 cc., and continuously extracted with ether for 8 hours. The evaporation with hydrochloric acid might be expected to hydro- lyze methyl oxaluric acid (17), but methyl parabanic acid is very stable toward acids. A sample was prepared by the method of boiling theobromine with sulfuric acid and potassium bichromate and extracting with ether. The acid gives no precipitate on adding calcium chloride to its solution acidified with hydrochloric acid, and neutralizing with potassium acetate. Oxalic acid was precipitated quantitatively under these conditions. And when 10 cc. of 0.02 M parabanic acid solution were made alkaline with 2

by guest on June 23, 2020http://w

ww

.jbc.org/D

ownloaded from

0. H. Gaebler and A. K. Keltch

cc. of 10 per cent sodium hydroxide and boiled 2 or 3 minutes, the calcium oxalate precipitated after adding calcium chloride solution and acidifying with acetic acid required 3.80 cc. of 0.1 N

permanganate (theory 4.0). The ether extracts from urines were therefore evaporated over 10 cc. of water, made up to 50 cc., and filtered. Such filtrates are always acid. 20 cc. portions were treated with calcium chloride and potassium acetate directly, and separate 20 cc. portions were boiled with alkali as above, and,

TABLE V.

Date.

1996

Jan. 4 “ 5 “ 6

gL 7 Ld 8 “ 9

“ 10 “ 11

-

-

2.7C 2.71 3.6C

2.8: 2.9c

Pi 2 4 ;: i g 4 s -- 07% gm. 0.21 2.24 0.22 2.OC 0.09 2.3:

m' .B

2.2 .; 2.i I

.@ E$ G 0 --

no. no. 234 28 242 29 245 18

0.23 1.9( 222 16 0.22 2.0: 244 16 0.39 2.0: 284 91

0.15 2.2: 0.18 2.3i

228 4 220 6 ____-

D og I. -

--

-

mo. 14.: 7.2

11.2

4.E 15.4 1l.f

11.6 10.4

cc

, 1

!

;

i I

I

-

:. 0.1

116 115 376

109 120 292

122 120

-

-_

N

-

Remarks.

4.15 gm. hydantoic acid: neutralized, in three doses subcutaneously,

3.5 gm. hydantoin in three doses subcu- taneously,

after addition of calcium chloride, acidified with acetic acid. The values for free and total oxalic acid are given in Table IV.

Experiment 5 (Table V).-The same animal was used, but now weighed 12 kilos. The diet was the same as in Experiment 2. Hydantoin and sodium hydantoate were used in this experiment The unchanged hydantoin can be calorimetrically estimated by the same method used for methyl hydantoin. On January 6 4.15 gm. of hydantoic acid were given subcutaneously, after neu- tralizing with sodium hydroxide. There was a fall in ammonia and rise in urea nitrogen, which may both have been related to increased amount of available base due to the administration of

by guest on June 23, 2020http://w

ww

.jbc.org/D

ownloaded from

770 Metabolism of Hydantoins

sodium salt. The rise in organic acids is 260 cc. 0.1 N per day. On adding 0.415 gm. of the acid to one-tenth the urine of January $, and repeating the determination, the organic acid figure changed from 120 cc. 0.1 N to 452 cc. 0.1 N, -a rise of 332 cc. 0.1 N (theory 350 cc.). On January 9, 3.50 gm. of hydantoin were given sub- cutaneously in three doses. As in the case of sodium hydantoate the total nitrogen excreted accounted for all of the compound given. There was a rise in ammonia. The color curve of the urine with alkaline picrate could be duplicated by adding the equivalent of only half the hydantoin administered to a urine of a control day, and organic acid showed a rise which would account for the other half of the compound as hydantoic acid. Benzal hydantoin (8, 18), isolated from the urine of January 6 after evaporation with strong HCl, amounted, after recrystallization from alcohol, to 1.34 gm. from half of the day’s urine, or the equivalent of 40.9 per cent of the hydantoic acid given (N, 14.81 per cent; m.p. 217.5-218.5”). From half of the urine of January 9, after evaporation with HCl, there were obtained 1.32 gm. of recrystallized benzal hydantoin, or the equivalent of 40.1 per cent of the hydantoin given (N, 14.82 per cent; m.p. 217.5-219”). Wheeler and Hoffman (18) obtained 70 to 80 per cent yields, appar- ently not recrystallized, when working with pure hydantoin in amounts five times as great. The above isolation would therefore scarcely be expected had the hydantoin not excreted unchanged been excreted as any compound other than hydantoic acid.

Experiment 6 (Table VI) .-A different animal was used in this experiment,-an adult bitch, weighing 11 kilos. The diet was the same as in Experiment 2, excepting that phosphoric acid and acid phosphates were added to the diet to keep the urine acid. The animal was pregnant and gave birth to a litter of six healthy pup- pies 3 weeks after the close of the experiment.

On May 28,4.65 gm. of methyl hydantoic acid, neutralized with sodium hydroxide, were given subcutaneously in three doses. There was a decided rise in urea nitrogen, but the rise in total nitrogen exceeded that of the compound given (0.98 gm.) by 0.35 gm., taking the average of the preceding and following day as con- trol. Urea nitrogen, however, rose 0.70 gm. On June 2, 4 gm. of methyl hydantoin were given, but the last dose was not adminis- tered until 11.40 p.m., so that the excretion carries over into the

by guest on June 23, 2020http://w

ww

.jbc.org/D

ownloaded from

0. H. Gaebler and A. K. Keltch 771

following day’s urine, which began at 9 a.m. The rise in total nitrogen for June 2 and 3 is 0.29 gm. more than the nitrogen of the compound, and the rise in urea nitrogen is 0.66 gm. The amount of methyl hydantoic acid excreted unchanged on May

TABLE VI.

Dog 2.

----

19.26 gm. gm. gm. mg.

May 26 3.790.24,2.97284 “ 27 “ 28

“ 29 ‘[ 30 “ 31

.hne 1 “ 2

“ 3 (‘ 4 “ 5 “ 6 “ 7

“ 8 2.620.241.92228 2.620.241.92228

13.6510.3312.651280 3.650.332.65280 4.85 0.22 3.33 280 4.85 0.22 3.33 280

i .‘: 0 z

0 -

w.

16 5

25

a 2 % o 4 s * 2 2 .-

s rd 0” 5 2 8: -F; $ & g &

--- w?. mQ.

cc. 0.1 N

16.3 18.5 11.2 12.3 84.5 146.0

I 1 I I 3.400.232.61288 3.400.232.61288 10 32.6 50.0

3.140.322.15264 3.140.322.15264 28 10.7 14.1 4.000.362.42283 4.000.362.42283 21 .21.0 184.0

3.53 0.37 2.56 264 3.53 0.37 2.56 264 3.120.342.16245 3.120.342.16245

2.640.29 1.84;222 2.64 0.29’1.841222 3.65 0.20 2.16:298 3.65 0.20 2.16’298

10 8

8

-

70.5 129.5 19.7 27.6

15.2 20.2 96 20.2 29.2 300

16.9 24.2 124 6.01

-

I Remarks.

x a -

5.9 6.3 5.8 4.65 gm. methyl:

hydantoic acid, neutralized, in three doses sub- cutaneously.

6.1

7.0 7.0 4.0 gm. methyl

hydantoin in three doses sub- cutaneously.

6.8 7.0

6.7 5.8 3.5 gm. hydantoin

in three doses subcutaneously.

28 was estimated calorimetrically as 56 per cent of that given. Isolation as methyl hydantoin was accomplished by evaporating the urine with hydrochloric acid, continuously extracting with chloroform, evaporating the chloroform, and recrystallizing the residue from alcohol. The recrystallized product was still slightly

by guest on June 23, 2020http://w

ww

.jbc.org/D

ownloaded from

772 Metabolism of Hydantoins

brown. Nitrogen content, 23.9 per cent (theory 24.6). M.p. 152-155.5”, uncorrected (accepted 156’, corrected). The solubili- ties and color reaction were those of methyl hydantoin. The yield, of recrystallized product, calculated to 24 hours, was 0.985 gm. (as methyl hydantoin) or 24.6 per cent of the acid given. The .color curve of the unhydrolyzed urine with alkaline picrate showed a very slight rise during the 1st and 2nd hour, indicating a possible trace of methyl hydantoin. The amount of methyl hydantoin excreted unchanged on June 2 and 3 was estimated lcolorimetrically as 24 per cent of that given, and the “total methyl hydantoin” as 32 per cent. On June 7, 3.5 gm. of unsubstituted hydantoin were given. The results were identical with those in Experiment 5. Calorimetric determination indicated excretion of slightly less than one-half of the compound unchanged, and the rise in organic acids would account for a little more than half of the compound as hydantoic acid. Isolation of benzal hydantoin by a slightly modified method, from half of the urine, yielded 1.81 gm., or 55 per cent of theory (m.p., 217.5’;N, 14.5 per cent). By the same method the isolation of benzal hydantoin from half of a 24 hour urine to which half the amount of hydantoin adminis- tered was added, amounted to 67 per cent of the theoretical.

The marked decrease in total nitrogen on the constant diet was chiefly an adaptation to low protein intake, perhaps accentuated by pregnancy. The animal also shows the known decrease in ereatinine as the result of cage life to a remarkable degree. The blood urea nitrogen amounted to 14.3 mg. per 100 cc., and “creat- inine” 1.5 mg. per 100 cc. at the close of the experiment.

Experiment 7 (Table VII.)-Dog 2 was used again in this experiment, after it had weaned its puppies. The daily ration consisted of 150 gm. of a mixture prepared by grinding together 800 gm. of soda crackers and 700 gm. of round steak. 30 cc, of a normal solution of acid sodium phosphate were added to each day’s ration. The oxidation of methyl hydantoin in this as in the preceding experiment is smaller than in the case of Dog 1. Urea determinations by the Benedict method, as in Experiment 2, Table II, account for little more, if any, of the extra nitrogen on experimental days than determinations by urease. Methyl urea added to control urines appeared almost quantitatively as a difference between urea determinations by these two methods.

by guest on June 23, 2020http://w

ww

.jbc.org/D

ownloaded from

0. H. Gaebler and A. K. Keltch 773

Methyl amine would also appear in this way, since the ammonia determinations subtracted from the Benedict ureadetermination were done by means of permutit and Nessler’s reagent, whiIe the urea plus ammonia nitrogen in the Benedict method is obtained by distillation and titration. While methyl amine is removed by permutit (19) we find that it gives a coIor entirely comparable with that of ammonia, but only a fifth as great per equivalent of nitrogen. Ethyl amine gives a color one-third as great as that of ammonia, while dimethyl, trimethyl, diethyl, and propyl amines give no color nor yield precipitates. Erdmann (20), no doubt using

TABLE VII.

Dog 2.

Date.

19?36

Aug. 16 “ 17

“ 18 “ 19 “ 20

“ 21

2 ti .$z P 2 s 4 -- Q+% gm. 3.02 O.l( 3.87 0.1:

_-

-

3.08 0.1: 2.95 O.O! 4.01 O.O!

2.66 0.0’

m a --- gm. llm. m0. m3.

2.37 2.34 14. 19.! 6.6 2.44 2.31128. 193.t 6.3 4.0 gm. methyl hydan-

toin in three doses subcutaneously.

2.48 2.40 50.’ 74.: 6.6 2.39 2.28 15.’ 17.1 6.4 2.60 2.40 102. 152.t 6.8 4.65 gm. methyl hydan-

toic acid, neutral- ized, in three doses, subcutaneously.

2.091 1.951 10. 48.: 6.5

-

3 3

2 a a

9 -

Remarks.

_ .

8 51

7 2 31

6 -

a different Nessler formula, states that amines give lemon-yellow precipitates with the reagent.

Methyl parabanic acid is unaffected in the Benedict urea de- termination. While it would be decomposed into methyl urea and oxalic acid during the distillation, distillation of methyl urea with the amount of alkali used, not preceded by digestion with potassium acid sulfate and zinc sulfate, yields little nitrogen.

The isolation of the “combined” oxalic acid was attempted. One-half of the urine of each of the experimental days was evapo- rated to small volume with 12.5 cc. of concentrated HCI, and con-

-

by guest on June 23, 2020http://w

ww

.jbc.org/D

ownloaded from

774 Metabolism of Hydantoins

tinuously extracted with ether. The ether extract was evaporated and sublimed at 135”. Oxidizing acids must be absent in such a sublimation, since unchanged methyl hydantoin otherwise yields methyl parabanic acid. A considerable sublimate of needles was obtained in each case. These sublimates consisted largely of methyl hydantoin. Solutions of the same gave no precipitate with calcium chloride and potassium acetate, but after boiling with alkali, adding calcium chloride, and acidifying with acetic acid, a white precipitate, insoluble in water and titratable with permanganate to a sharp end-point, was obtained. The amount was very small, corresponding to 7 mg. of methyl parabanic acid per day. It was found, however, that of 50 mg. of methyl parabanic acid added to a similar amount of a control urine, only 5.5 mg. were recovered by this procedure. In Experiment 7 only about 70 mg. of methyl parabanic acid could have been excreted per day, hence the result is not surprising.

Although none of the urines showed evidence of cystitis, such as putrefaction or turbidity which would not clear on addition of acid, many of them had been retained 24 hours by the animals. The effect of incubation was therefore studied. Portions equal to one-fourth of the day’s urine, from several control days, were incubated with one-fourth the amount of methyl hydantoin given, for 24 hours at 4O”C., with and without toluene. The urines were then acidified, concentrated, and extracted as usual. In absence of toluene, the urine became turbid from bacterial growth, and neutral or alkaline. Neither free nor combined oxalic acid could be recovered.

The urine incubated with toluene remained clear and acid, and the values for free and total oxalic acid were those of the original control urine. A previous experiment in which 125 cc. of urine (one-fourth of the daily output) were allowed to stand under toluene at room temperature, after addition of 1 gm. of methyl hydantoin, gave similar results. Calorimetrically determined, the methyl hydantoin fell to about 75 per cent of its original .value in several weeks. No increase in oxalic acid could be determined, but much methyl hydantoin could be isolated.

by guest on June 23, 2020http://w

ww

.jbc.org/D

ownloaded from

0. H. Gaebler and A. K. Keltch 775

DISCUSSION.

The chief points of interest in the preceding experiments are the small percentage of methyl hydantoin which is excreted entirely unchanged and the large amounts of free and combined oxalic acid which appear. The latter effect appears much too great to be interpreted as anything but oxidation of the compound given. The failure of such oxidation in the absence of the methyl group is also clear cut.

It is well known that oxalic acid may be destroyed in the body (13), although Wegrzynowski (21) appears to regard the small normal excretion as evidence of very limited formation. The amount of oxalic acid that was excreted in our experiments might only represent a fraction of the extent of actual oxidationofmethyl hydantoin. The failure to obtain evidence for the excretion of a great deal of methyl urea (Experiments 2 and 7) does not suggest a much more extensive oxidation than oxalic acid determinations w,arrant, at least by the two courses of oxidation which we have considered (22). It does not, however, preclude the possibility that the rise in free oxalic acid might be due to decomposition of methyl parabanic acid. The largest amount of total oxalic acid excreted (Table III) was 0.355 gm., which corresponds to 0.45 gm. of methyl hydantoin and 0.11 gm. of nitrogen. The mentioned possibility also is not excluded by the fact that the 24 hour urines were acid, since the urine as secreted might not always be acid.

The two courses of oxidation considered were: (1) transforma- tion of methyl hydantoin to methyl hydantoic acid, with hydroly- sis of the latter to methyl urea and glycollic acid, followed by oxidation of glycollic to oxalic acid, and (2) oxidation of methyl hydantoin to methyl parabanic acid. The amount of total oxabe acid excreted on experimental and following days was 30 to 60 per cent greater (Tables VI and VII) when methyl hydantoin was given than when the corresponding amount of methyl hydantoic acid was administered. This may be interpreted as indicating that the acid must be transformed to the hydantoin, previous to oxids tion, but is not very conclusive since excretion rates may account for the observation. The only direct evidence for such a course which we have is the observation that urines of August 17 and 20, Table VII, both contained a combined form of oxalic acid which

by guest on June 23, 2020http://w

ww

.jbc.org/D

ownloaded from

776 Metabolism of Hydantoins

resisted evaporation with hydrochloric acid, was easily extracted with ether and sublimed. Methyl parabanic acid is known to fulfil these requirements, while one would not expect such behavior of methyl oxaluric acid, or dehydration of the same to methyl parabanic acid.

The nitrogen, of the administered compounds, which in al1 cases, excepting Table I, equals 0.98 gm., is more completely accounted for in the case of methyl hydantoic acid than in the case of methyl hydantoin. In Experiment 6, Table VI, 56 per cent of the methyl hydantoic acid given was excreted unchanged. The rise in urea nitrogen both in this and the more favorable ex- periment recorded in Table VII accounts for an additional 12 to 20 per cent. In the case of methyl hydantoin from 30 to 50 per cent was excreted either as such or as methyl hydantoic acid, while irregular rises of ammonia and urea varied from 20 per cent of the nitrogen of the compound to zero. About one-half of the extra nitrogen excreted as the result of methyl hydantoin administra- tion was therefore unidentified.

CONCLUSIONS.

1. A considerable portion of methyl hydantoin and methyl hydantoic acid administered to dogs subcutaneously is oxidized, probably to methyl parabanic acid.

2. The oxidation fails in the absence of the methyl group. 3. Both in the case of methyl hydantoin and of unsubstituted

hydantoin, a fraction is excreted unchanged, and another fraction as the corresponding acid.

BIBLIOGRAPHY.

1. Schultzen, O., Ber. them. Ges., 1872, v, 578. 2. Baumann, E., andHoppe-Seyler, F., Ber. ehem. Ges., 1874, vii, 34. 3. Baumann, E., Ber. them. Ges., 1874, vii, 237. 4. Baumann, E., and van Mering, J., Ber. them. Ges., 1875, viii, 584. 5. Schiffer, J., Z. physiol. Chem., 1881, v, 257. 6. Lippich, F., Ber. them. Ges., 1908, xli, 2974. 7. Dakin, H. D., J. Biol. Chem., 1910, viii, 25. 8. Lewis, H. B., J. Biol. Chem., 1912-13, xiii, 347. 9. Folin, O., and Bell, R. D., J. Biol. Chem., 1917, xxix, 329.

10. Van Slyke, D. D., and Cullen, G. E., J. Biol. Chem., 1914, xix, 211. 11. Folin, O., J. Biol. Chem., 1914, xvii, 473. 12. Wiechowski, W., Beitr. them. Physiol. u. Path., 1968, xi, 109.

by guest on June 23, 2020http://w

ww

.jbc.org/D

ownloaded from

0. H. Gaebler and A. K. Keltch 777

13. D&in, H. D., J. Sol. Chem., 1907, iii, 57. 14. Van Slyke, D. D., and Palmer, W. W., J. Biol. Chem., 1920, xii, 567. 1.S. Ellinger, A., and Matsuoka, Z., 2. physiol. Chem., 1914, lssxix, 441. 16. Gaebler, 0. H., J. Biol. Cheer., 1926, lxix, 613. 17. Behrend, R., and Dietrich, IS., Ann. Chenz., 1899, ceeix, 271. 18. Wheeler, H. L., andHoffman, C., Am. Chem. J., 1911, xlv, 368. 19. Vhitehorn, J. C., J. Biol. Chem., 1923, Ivi, 751. 20. Erdmann, C. C., J. Biol. Chcm., 1910, viii, 41. 21. Wegrzynowski, L., Z. ph?ysioZ. Chem., 1913, lxxxiii, 112. 22. Gaebler, 0. I-I., J. Biol. Chcm., 1926, lxvii, p. Iv; Proc. Sot. Ezp. BioZ-

and Med., 1926, xxiii, 479.

by guest on June 23, 2020http://w

ww

.jbc.org/D

ownloaded from

Oliver Henry Gaebler and Anna K. KeltchACIDS

HYDANTOINS AND HYDANTOIC ON THE METABOLISM OF

1926, 70:763-777.J. Biol. Chem. 

  http://www.jbc.org/content/70/3/763.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

alerts to choose from all of JBC's e-mailClick here

  ml#ref-list-1

http://www.jbc.org/content/70/3/763.citation.full.htaccessed free atThis article cites 0 references, 0 of which can be

by guest on June 23, 2020http://w

ww

.jbc.org/D

ownloaded from