ON THE MAXIMUM PRODUCTION OF HIPPURIC ACID IN ANIMALS WITH CONSIDERATION OF THE ORIGIN OF GLYCOCOLL IN THE ANIMAL BODY.

BY A. I. RINGER.

(From the Physiological Laboratory of the Cornell University Medical College, New York City.)

(Received for publication, September 9, 1911.)

INTRODUCTION.

Parker and Luskl fed lithium benzoate to rabbits, with the object of ascertaining how much glycocoll the animals mayeliminate in the formation of hippuric acid. They also studied the relation- ship between the total nitrogen in the urine and the glycocoll

(H.A.N). nitrogen eliminated as hippuric acid ~

N They found that

for every 100 grams of total nitrogen excreted in the urine, an average of 4 grams of nitrogen was eliminated in the glycocoll radical of the hippuric acid. After administering the first large dose of lithium benzoate to the animals, they invariably obtained

a much higher H* ^,’ N’ ratio; 9.01 per cent in experiment III, 7.14

per cent in experiment IV and 7,87 per cent in experiment VI. They regarded this as a “sweeping out of the surplus glycocoll” (in the sense of Wiene?).

Since then Wiechowsk? and Magnus-Levy4 simultaneously, but independently, have studied the same problem. Wiechow-

H.A.N. ski obtained -

N ratios of 45.4, 55, 50, and in one case even as

1 Amer. Journ. of Physiol., iii, p. 472, 1900. ‘Arch. j. exp. Path. U. Pharm., xl, p. 313, 1898. * Beitr. z. them. Path. u. Physiol., vii, p. 204, 1906. ‘Biochem. Zeitschr., vi, p. 523, 1907.

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Production of Hippuric Acid

much as 64.3 per cent. Magnus-Levy found in his rabbits a max- imal ratio of 25 and 28 per cent; in his sheep 27.8 per cent.

The various forms of animal protein, with the exception of casein which on analysis yields no glycocoll, and gelatin which yields 16 per cent, contain on an average 4 per cent of glycocoll. Since the nitrogen content of protein is 16 per cent and that of glycocoll 18.7 per cent, 100 grams of protein nitrogen therefore contain 100 X 18.7 X 4

16 = 4.7 grams of preformed glycocoll nitrogen. Mag-

nus-Levy found that after feeding large doses of benzoic acid to rabbits and sheep, they were able to yield six times that amount of glycocoll. Wiechowski’s rabbits yielded almost fourteen times that amount. These findings stand in direct contradiction to the conclusions arrived at by Parker and Lusk. To investigate the cause of these discrepancies, Professor Lusk kindly suggested that I continue the study of this problem. It may be added here, that Parker and Lusk were influenced by the rather inaccurate statement of Wiener, that 1.7 grams of benzoic acid per kiiogram of body weight administered to rabbits was fatally toxic.

METHODS OF EXPERIMENTATION.

The animals were kept in metabolism cages. The urine was collected by catheter and separated into twenty-four-hour periods. Sodium benzoate dissolved in water was given per OS by means of a stomach tube. The daily dose was always divided into three to five doses, given five to three hours apart. The animals were catheterized before the stomach tube was introduced, so as to prevent any accidental loss of urine.

The total nitrogen was determined in duplicate by the ordinary Kjel- dahl method. The hippuric acid, also in duplicate, was determined by the method proposed by Dakin.1 One-quarter of the total quantity of urine was evaporated on the water bath to about 75 cc. After transfer- ring to a small Erlenmeyer flask, 15 cc. of a 50 per cent solution of phos- phoric acid was added. The mixture was then cooled in running tap water. The greater amount of hippuric acid then crystallized out. The solution was filtered and the crystals washed with a small quantity of ice water (Filtrate A). The crystals were then dissolved in a rather large quantity of warm water, transferred to a separatory funnel, and shaken with about

‘This Journal, vii, p. 103, 1910.

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A. I. Ringer

50 to 100 cc. of amixture of 2 parts of benzol and 1 part of alcohol-free ether. This removed any free benzoic acid which might have come down with the hippuric acid. After drawing off the water solution of the hippuric acid, it was slowly evaporated and crystallized. The crystals were separated by filtration and washed with a small quantity of cold water (Filtrate B). The crystals were dried and weighed in the usual manner. (Gooch crucibles were found to be very convenient receptacles for the crystals.)

To Filtrate A about 75 per cent of its weight of ammonium sulphate’ was added. It was then placed in a continuous extraction apparatus and extracted with ethyl acetate for a period of eight to ten hours.

The ethyl acetate was then drawn off into a separatory funnel and shaken with a small quantity of a saturated sodium chloride solution. This removed any urea which might have been extracted by the ethyl acetate. The salt solution was then drawn off, and the ethyl acetate washed with a few cubic centimeters of distilled water to remove the crystals of sodium chloride which remained attached to the funnel. The salt solution, which had been drawn off, was then shaken with some ethyl acetate, which was finally added to the original.

The combined ethyl acetate solutions were then distilled in c’ocuo and the residue dissolved in hot water. This was boiled with asmall quantity of blood charcoal to decolorize it, filtered, and the charcoal washed with a small quantity of boiling hot water. After the filtrate had cooled,it was placed in a separatory funnel and shaken four to five times with about 100 cc. of the benzol and ether mixture. The aqueous solution of hippuric acid was then drawn off, and Filtrate B added to it. The combined solu- tion was allowed to evaporate slowly and the hippuric acid which crys- tallized was purified, dried and weighed in the usual manner.

The sum* of all the crystals obtained, multiplied by four, gave the value of hippuric acid excreted during the course of the period.

CONTROL OF THE METHOD.

To 100 cc. of normal human urine 1 gram of hippuric acid (Kahlbaum) was added. Recovered from the first crystallization, 0.3034; from the second, 0.1173; from the third, 0.0580 gram. Total=0.9787 gram, or 97.87 per cent.

The melting point of the crystals was determined in every case. It was found to be between 182O and 187’ C. (uncorrected). The nitrogen content of the crystals was found to be between 7.94 and 7.99 per cent.

‘The ammonium sulphate serves a double purpose. First, it increases the rate of extraction of the hippuric acid. Second, it prevents the solu- tion of the ethyl acetate by the water, which would obliterate the line of demarcation between the watery solution and the ethyl acetate.

*Not all the hippuric acid crystallized out from the mother liquid with the first crystallization. Two, and sometimes three crystallizations were required to bring them all down.

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330 Production of Hippuric Acid

The duplicates of the hippuric acid determinations agreed within 0.3 per cent.

EXPERIMENT I.

A goat weighing 42.3 Kg. was employed. She was brought to the laboratory about a week prior to the commencement of the experiment and was placed in a metabolism cage. During the foreperiod, which lasted for three days, the goat was fed on hay, cornmeal and oats. Only the total nitrogen eliminated in the urine was studied.

During the benzoate period the diet could not be regulated very well. She was fed on hay, bread, carrots, cabbage and milk, the daily quantity of which is recorded in Table I. During the peri- ods VI, VII and VIII, she did not take any food.

During the first day of the benzoate period, the animal received 10 grams of sodium benzoate, the second, 20 grams, the third, 20 grams, the fourth, 25 grams, the fifth, 10 grams, the sixth, 30 grams, the seventh, 30 grams. On the eighth day she also received 30 grams, but all in one dose. At the end of that day the animal was still in very good condition and showed no signs of intoxica- tion.

After sixteen days intermission an attempt was made to dis- cover a dose which would produce toxic symptoms in the animal. To this end the animal was given 50 grams of sodium benzoate in three equal portions during the course of the day (period IX). On the following morning, distinct signs of intoxication were pres- ent. The animal was exceedingly restless. She ran about the cage, striking her horns violently against the walls. She partook of neither food nor drink. When allowed to walk out of the cage, she did not walk in a straight line, but in a zigzag manner. When she struck the wall of the room, she did not turn backwards, but kept on pushing in the same direction. In walking she did not avoid any object placed in her way. She gave the impression of having lost the power of seeing. It is to be regretted that the reflex of the eye towards light was not tested. These symptoms persisted until the following morning, when the animal was seized by an attack of tonic and clonic convulsions in which she died.

Autopsy revealed no macroscopical changes in the organs. She was very much emaciated. On section the kidneys showed slight parenchymatous tubular degeneration.

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TABL

E I.

Goa

t I.

DATE

. 19

10

z RE

FdbB

K.9

8 z F?

4

i f

E PI

yy

p~-_

_---

gram

s pr

om*

gram

s ip

rree

nt

per

cent

Fe

brua

ry

25...

. .

. ?

Febr

uary

26

.. . .

.

Febr

uary

27

.. .

. .

c(

Febr

uary

28

.. . .

. .

* M

arch

1.

. .

. .

. .

II U

rine

lost

F*

19

0 gr

ams

milk

. M

arch

2

. . .

. . .

. . .

. 20

0 gr

ams

whi

te

brea

d 15

0 gr

ams

hay.

(D

&

Mar

ch

3..

. .

. 10

0 gr

ams

brea

d,

lOO

gram

s ca

bbag

e,

rt 16

0 gr

ams

carr

ots.

M

arch

4.

. . .

. .

. V

40

.1

6.45

0 11

.06

0.87

4 13

. A

te

wha

t w

as l

eft

from

pr

evio

us

day

and

no

mor

e.

Mar

ch

5..

. . .

. . .

VI

6.

360

30.9

3 2.

443

Mar

ch

6 . .

. . .

. . .

. .

VII

39.3

5 8.

060

28.2

8 2.

234

Mar

ch

7..

. .

. .

VIII

one

dose

.

Mar

ch

23..

. .

IX

I 8.

1201

19

.96

1.57

71

19.4

1;

42.3

61

13.6

1 32

.15

Sta

rvin

g.

: H

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33’ Production of Hippuric Acid

Discussion of Results of Experiment I. The results obtained from the first experiment are summarized in Table I. It shows that the animal was able to eliminate in its urine a good deal more gly- cocoll than is found preformed in the proteins of its tissues. The hippuric acid formation, i.e., the glycocoll elimination, does not depend upon the amount of the protein catabolized, but within certain limits, runs parallel to the amount of benzoic acid fed. In period VI it reached its maximum, when 38.4 per cent of the total nitrogen was eliminated as glycocoll nitrogen in the hippuric acid.

Where do the enormous quantities of glycocoll originate? There are a number of possibilities which have to be taken into consider- ation.

It might be argued, since herbivorous animals have the power of eliminating more glycocoll than do carnivorous animals, when given a similar quantity of benzoate, that the source of the gly- cocoll found in the system lies in the different vegetable foods which are known to contain rather large quantities of free amino bodies. The results of experiment II, however, show that this assumption is not well founded.

EXPERIMENT II.

A fifteen day old calf, which had not yet received any other form of food but its mother’s milk, the protein of which contains practically no glycocoll, was placed in a metabolism cage, and its urine was collected for about twelve hours. On the following morn- ing, the calf received 8 grams of sodium benzoate per OS. Its urine was then collected for six hours.1

TABLE II.

Calf.

March 19 . . . . . . . . . . . . . . March 20. . . . . . . . . . . . . .

‘The catheter was not used. Hence the results are only approximate

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A. I. Ringer 333

We see from Table II that the suckling calf yielded hippuric acid as readily as would an adult goat. The glycocoll in the hip- puric acid could not have come from any other source than from a specialized protein metabolism.

Knoopl fed cu-ketonic acids to animals and obtained the corre- sponding amino-acids, showing that amination of non-nitrogenous substances can take place in the animal organism.

? P co > CHNH%

I I COOK COOH

a-Ketonic acids, together with the corresponding oxy-acids, can be found in the organism as a result of the oxidation of the a-amino bodies, or from the breaking down of carbohydrates.2 These facts raised the probability that glycocoll may be synthe- sized in the animal organism from non-nitrogenous substances chemically related to it.

CHs OH CH* NH* COH

I > I - I COOH COOH COOH

Glycollic Acid. Glycocoll. Glyoxylic Acid.

The experiments* recorded below were performed with the object of testing the truth of this theory.

In Table III the results of experiment III are summarized. On the first day the animal received, per OS, 4.5 grams of sodium ben- zoate dissolved in warm water. The dose was divided into four equal portions, given about three hours apart. On the second day the animal received 7.5 grams of sodium benzoate and 3

‘Zeitschr. f. physiol. Chem., lxvii, p. 491, 1910; Deutech. med. Wochen- schr., xxxvi, p. 1432, 1910.

‘Riihman: Biochemie, p. 540. *Experiments III, IV and V were performed in the laboratories of the

Second Medical Clinic of the University of Munich. I wish to express my thanks to Prof. Friedrich von Miiller for his kindness in granting me the freedom of his laboratory.

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DbTE

, 19

10

June

9.

....

.....

June

10

.....

.....

-

t 1 3.53

3.48

- -- gr

ama

2.67

3.47

5.

64

TABLE

III.

Rab

bit

III.

0.44

6 12

.8

3.01

3.85

_-

60.6

TABLE

IV

.

Rab

bit

V.

1.25

1.62

BE

?.M

BK

s cp

z

4 X

1.12

5 of

so

dium

be

nsoa

te

+ 2

gram

s o

300

cc.

crea

m

give

n pe

r O

S.

I-.

5 X

1.5

sodi

um

benz

oate

+

3 gr

ams

gram

s 2

glyc

ollic

ac

id

+ 30

0 cc

. cr

eam

. 0 ; %

July

19

.. . .

. Ju

ly

20..

. . . .

. . . .

..I .

2.61

.

i 0.

975

) 1.

626

3.56

)

0.28

1 1

17.3

1

3.39

i

2.42

i

71.3

1

1.37

1

Amm

al Aq

ima1

st

ar’in

g*

star

ving

. 4g

ram

sofs

odiu

mbe

nzo-

at

e +

2 gl

ycol

lic

acid

gi

ven

OS.

gr

ams

per

~ C.

CL

TABLE

V

.

Rab

bit

IV.

June

29

Ju

ne

30 . .

. . . . . . . . . . .

. . . . . .

..I .

3.76

3.

63

3.39

66

.7

0.91

St

arvm

g.

Sta

rvjn

g.

4gra

mso

fsod

ium

benz

oate

pero

s.

July

1.

. .

. .

3.41

Il.339

1 2.

05

2.59

3.31

7 1

6.48

8

0.26

2 1

0.51

2

12.7

8 )

19.8

5.

08

/ 2.

26

4.42

)

87.0

1.

90

1

Sta

rvin

g.

6 so

dium

be

nzoa

te.

gram

s __

_--

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A. I. Ringer 335

grams of glycollic acid equally distributed into five d0ses.l (The glycollic acid had been neutralized by means of sodium bicarbon- ate.) The result was an increase of 1.2 grams of hippuric acid over the amount in the previous period. As is seen from Table V, this increase in the hippuric acid elimination cannot be attrib- uted to the introduction of the glycollic acid, but to the larger dose of benzoic acid. The administration of larger doses of benzoic acid, if also accompanied by a simultaneous administration of glycollic acid, was followed by fatal results (Rabbits I and II).

It was observed in experiments I, II and III that not all the benzoic acid administered was eliminated as hippuric acid. This phenomenon was explained by previous authors as being due to a lack of glycocoll in the system. If this were true, and if glycol- lit acid actually did go over into glycocoll, then we would expect, on feeding glycollic acid + the benzoate, that the relationship

Benzoic acid fed

Benzoic acid excreted as hippuric acid

would approach 100 per cent. But the results in experiment IV

(see Table IV) show that this is not the case. The t ratio

remains as low as in the other experiments. In summing up the results of the last three experiments, we do

not find any reasonable ground for the assumption that the gly- cocoll is synthesized from the non-nitrogenous glycollic acid.

On careful consideration, however, of the nitrogen metabolism in the animals during the benzoate period, a possible explanation of the origin of glycocoll suggests itself. The goat, during the three days of the foreperiod, excreted an average of 5.6 grams of nitrogen per day. Excepting the first day, which may be due to a sudden change in the quantity of food, there is a marked rise in the protein metabolism throughout the course of the benzoate period. This was observed in all the experiments and in all the varieties of animals that were experimented upon, Furthermore, the increase in the protein destruction, i.e., the increase in the nitrogen elimination above the normal, or above that of a pre-

‘Rabbits I and II were treated in a similar manner. Both died during the course of the second day, owing to the large dose of benzoate. Rab- bit III died on the day following the experiment. Rabbit IV lived for two days after the experiment. Rabbit V survived.

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336 Production of Hippuric Acid

vious day of a smaller benzoate dose, is always two or three times greater than the amount of nitrogen that has been eliminated as glycocoll in the hippuric acid molecule.

Salkowskil on giving sodium benaoate, obtained an increase in the nitrogen elimination, accompanied by a corresponding increase in the sulnhur metabolism. In experiments VI, VII and VIII (see corresponding tables) we obtained a considerable increase. in the total nitrogen on the benzoate days, but the amount of urea remained practically the same as on the previous and following days. Similar results are said to have been obtained by Shepard and Meisner.2

All these facts suggest the possibility that the glycocoll excreted as hippuric acid did not come from the fraction of protein that would have been metabolized had no benzoate been given, but from the “extra protein” which was destroyed, due to the presence of the benzoic acid. We cannot state with any degree of certainty, what the character of the intermediary process is, but that it is specific and peculiar seems very probable, for none of the extra nitrogen went over into urea. It was all eliminated either as glycocoll or as undetermined nitrogen.

THE SYNTHESIS OF HIPPURIC ACID.

In the dog, the kidney is the only organ in which hippuric acid is synthesized.s In the rabbit it can take place even after nephrectomy4 and is probably brought about by the action of a ferment.

Not all of the benzoic acid fed is synthesized into hippuric acid, only an average of 78.1 per cent in the goat (excluding periods VIII and IX for reasons mentioned below) and 73 per cent in the rabbits. It is also to be noted that the relationship between the benzoate fed and the benzoate excreted in the hippuric acid mole- cule remains fairly constant, except when too large amounts of benzoate are thrown into the system at once, then it sinks, prob- ably because the cells of the body are able to synthesize a certain

1Virchow’s Archiu, lxxviii, p. 530, 1879. 2 Untersuchungen Qber das Entstehen der HippUTSdUTe, Hanover, 1866

(cited by Wiener, lot. cit.). 8Bunge and Schmiedeberg: Arch. f. exp. Path. u. Charm., vi, p. 233,

1877. 4Salomon: Zeitschr. f. physiol. Chem., iii, p. 365, 1879.

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TABL

E VI

.

Rab

bit

VI.

I I

-1

I - -

-

I l I

-

1 A a :!

gm?n

a 0.

802

1.14

7 0.

913

gram

s 0.

7024

D

.796

3.

782

I- ---

I-

f7sa

md

gram

s

87.6

0.

0232

2.

89

0.72

56

90.4

9 69

.4

0.03

6 3.

177

0.82

9 72

.58

85.6

7 0.

0166

1.

82

0.79

86

87.4

9

k&x

1.80

1.72

1.

60

Mar

ch

12 ..

......

. 1

Mar

ch

13 ..

......

. II

Mar

ch

14 ..

......

. II1

star

ving

. st

arvi

ng.

+ st

arvi

ng.

* c1

. w w..

star

ving

. 3

star

ving

. ‘t

star

ving

.

0.08

8 7.

7

TABLE

V

II.

Rab

bit

VIZ

.

81.6

8 0.

086

7.23

70

.66

0.11

27

8.23

TABLE

V

III.

Goz

t II.

1 -

Mar

ch

23 .

......

.. I

1.96

M

arch

24

....

.....

II 1.

71

Mar

ch

2.5

......

...

III

1.68

1.19

2 0.

974

1.36

9 0.

967

1.19

9

1.21

6 18

.7

4.29

7 87

.25

4.33

9 84

.19

4.84

7 74

.56

4.35

2 87

.02

-~

0.04

2 0.

85

0.05

3 1.

03

0.15

6 2.

4 0.

086

1.73

- -

May

23

....

......

. I

19.0

4.

925

May

24

....

......

. II

5.15

4 M

ay

25 ..

......

...

III

6.50

1 M

ay

26 .

......

....

IV

5.00

1 M

ay

27 .

......

....

V

4.47

8

4.25

5 86

.4

4.28

6 83

.16

4.69

1 72

.16

4.26

6 85

.29

Sta

rvin

g.

Sta

rvin

g.

Sta

rvin

g.

Sta

rvin

g.

Sta

rvin

g.

z u

12.7

5

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338 Production of Hippuric Acid

amount of benzoate when administered at a slow rate. If it is absorbed at a quicker rate than that at which the cells have the power of combining it with glycocoll, then the benzoate circulates in the blood as such. A good portion of it escapes through the kidney, while another portion is combined with glucuronic acid (compare period VIII of Table I with period VII).

The benzoate ,when present in the circulation in large quantities, probably also lowers the “hippuric acid synthesizing function” of the cells. This will explain why in period IX of Table I and in period II of Table III so small an amount of hippuric acid was

eliminated (low 2 ratio).

Magnus-Levy’s observation that starving animals eliminate less hippuric acid than do animals which are well nourished is not cor- roborated by the present findings.

In conclusion, I beg to express my thanks to Prof. Graham Lusk for suggesting this problem and for many valuable sugges- tions made during the course of the experiment.

CONCLUSIONS.

1. Goats and rabbits have the power of eliminating hippuric acid containing more glycocoll than is found preformed in the proteins they metabolize. As much as 38.4 per cent of the total nitrogen of the goat was eliminated as glycocoll nitrogen in the hippuric acid.

2. The ingestion of benzoate in large quantities results in a considerable increase in the nitrogen elimination. The produc- tion of urea is not affected by it.

3. The increase in the nitrogen elimination is much greater than the amount of nitrogen eliminated in the hippuric acid.

4. It is suggested that the large quantities of glycocoll originate from the “extra destroyed” protein, and not from the protein that would have been metabolized had no benzoate been given.

5. The diet has no influence on the amount of hippuric acid eliminated.

6. No synthetic production of glycocoll from glycollic acid could be determined.

7. A suckling calf, fifteen days old, which had never received the glycocoll complex in its food, was in full possession of the power to synthesize hippuric acid and eliminate it in large quantities.

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A. I. RingerGLYCOCOLL IN THE ANIMAL BODYCONSIDERATION OF THE ORIGIN OF

HIPPURIC ACID IN ANIMALS WITH ON THE MAXIMUM PRODUCTION OF

1911, 10:327-338.J. Biol. Chem. 

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