THE INFLUENCE OF PUTREFACTION PRODUCTS ON CELLULAR METABOLISM.

II. ON THE INFLUENCE OF PHENYLACETIC AND PHENYL- PROPIONIC ACIDS ON THE DISTRIBUTION OF

NITROGEN IN THE URINE.

BY YOSHIZUMI HIJIKATA.

(From the Medico-Chemical Institute, Kyoto Imperial University, Kyoto.)

(Received for publication, January 31, 1921.)

The investigations of H. and E. Salkowskil have established the fact that in the putrefaction of proteins, aside from other products, two are formed which are homologous with benzoic acid, namely, phenylacetic and phenylpropionic acids; and that these acids when introduced into mammals, reappear in the urine conjugated with glycocoll, the phenylacetic as phenaceturi@ and the phenylpro- pionic, having been oxidized to benzoic, as hippuric acid. It is possible, of course, that putrefaction of protein material in the intestinal canal may give rise to the acids investigated and thus account for the occurrence of phenaceturic and hippuric acids in normal urine. Unfortunately no attempts have thus far been successful in establishing directly the presence of either phenyl- acetic or phenylpropionic acid in the intestinal contents or excre-

1 Salkowski, E., 2. physiol. Chem., 1878-79, ii, 420; 1883-84, viii, 149; 1885, ix, 229, 491. Salkowski, E., and Salkowski, H., 2. physiol. Chem., 1882-83, vii, 161, 169; Ber. them. Ges., 1879, xii, 107.

2 Recently, Thierfelder and Sherwin reported that, after administering phenylacetic acid, there appeared in the urine of man, not phenylaceturic (as in the case of dogs and rabbits) or phenylacetornithuric acid (as with birds) but phenacetylglutamic acid which could be isolated, partly com- bined with urea and partly uncombined (Thierfelder, H., and Sherwin, C. P., Ber. them. Ges., 1914, xlvii, 2639-34; Jahresb. Thierchem., 1914, xliv, 1058; 2. physiol. Chem., 1915, xciv, 1).

141

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142 Cellular Metabolism

ment,3 possibly, owing to the difficulty of isolating small quantities of these acids from such mixtures.

Several studies have been made of the influence of the acids on the excretion of nitrogen in the urine. E. Salkowski4 has shown that, on feeding phenylacetic acid to rabbits, the nitrogen excreted in the urine is increased. Desgrez and Guende5 have reported that phenylpropionic acid causes a decrease in the nitrogen in the urine when fed to guinea pigs; the decrease being due to a smaller excre- tion of urea. This leads to the conclusion that two homologous aromatic acids, which probably arise from the putrefaction of protein material in the intestine and which can combine with the same nitrogen-containing conjugate, glycocoll, are playing quite opposing r8les in the excretion of nitrogen in the urine. If we assume with Salkowski and others? that the introduction of benzoic acid causes a marked increase in the decomposition of tissue pro- teins, the same effect must also be ascribed to phenylpropionic acid, since it is oxidized in the animal body to benzoic acid. Con- trary to Desgrez and Guende’s statement, therefore, introduction of the latter substance should cause an increase in thenitrogen excreted in the urine just as in the case of phenylacetic acid.

The assumption is justified by the results of the following experi- ments which were carried out in the manner described in an earlier paper. ’ A number of the rabbits used were maintained in N bal- ance by feeding ‘I tofukara, “8 while others were fasted. The total nitrogen was determined by the Kjeldahl method, the urea by means of urease, the ammonia by the method of Kruger, Reich, and Schittenhelm, and the amino-acids by Van Slyke’s method.

3 Tappeiner has shown the presence of phenylpropionic acid in the paunches of ruminants fed with hay; but it is uncertain whether the acid existed preformed in the hay or was formed in the alimentary canal from

protein (Tappeiner, H., 2. Bid., 1886, xxii, 23640). 4 Salkowski, E., Z. physiol. Chem., 1888, xii, 222. 5 Desgree and Guende, Compt. rend. Sot. biol., 1906, lviii, 526.

6 Salkowski, E., Z. physiol. Chem., 1877-78, i, 1; Virchows Arch. path. An.at., 1888, cxiii, 394. Virchow, Z. physiol. Chem., 1877-78, i, 78. Kum- agawa, M., Virchows Arch. path. Anat., 1888, cxiii, 134.

7 Hijikata, Y., Acta scholae medicinalis universitatis imperialis in

Kyoto, iv. 8 Tofukara, a waste product obtained in the preparation of bean cheese,

was pressed daily and a weighed quantity soaked in a fixed amount of water.

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Y. Hijikata 143

Experiments with Phenylacetic Acid.

Experiment I.-A 1,900 gm. rabbit, in N balance, received on Oct. 30, 0.5 gm. of the acid9 mixed with his food and on the following day, 1.0 gm. In both administrations the acid was neutralized with sodium carbonate solution.

On both days in which the acid was given in the food, the excre- tion of amino-acids increased strikingly, while that of the total nitrogen was unchanged, causing a considerable increase in the proportion of the former to the latter. The excretion of ammonia was unaltered. A small decrease in the ratio of urea to total nitrogen is mainly due to an insignificant decrease in the absolute quantity of urea. The fact that there was no increase in the total nitrogen excreted, was to be expected since the amount of acid administered was too small to cause any decomposition of tissue proteins.

Experiment I.-A rabbit, weighing 2,420 gm. and in N balance, received on both Nov. 13 and 14, by means of the stomach tube, 1.2 gm. of phenyl- acetic acid, dissolved in sodium carbonate solution.

It is apparent that on both days in which the acid was given, not only the output of amino-acids, but also those of total nitrogen, urea, and ammonia were increased, in spite of a considerable inter- ference with the appetite. While the relative quantity of urea was very slightly decreased, the relative quantities of ammonia and amino-acids were more than doubled. The experiment was ended when the animal refused to take any more food.

Experiment S.-The rabbit weighed 2,830 gm. Until fasting was begun, it received tofukara; then 35 cc. of water once daily through the stomach tube. On both the 4th and 5th days of fasting, it received 1.0 gm. of phenyl- acetic acid, neutralized with 35 cc. of sodium carbonate solution through the stomach tube.

The total nitrogen excretion decreased during fasting until the acid was administered. There was then an increase for 2 days

9 The phenylacetic acid was prepared by Totani, according to ~Mann, and was analytically pure. It was identical with that used in his experi- ments reported in his famous work, “nber das Verhalten der Phenylessig- s&ure im organismus des Huhns.” I take this occasion to express to him my thanks for his courtesy in supplying it (Tots& G., 2. physiol. Chem., 1910, lxviii, 75; Mann, W., Ber. them. Ges., 1881, xiv, 1645).

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-

Date.

Bo

dy

-i

weig

ht.

1

Tota

l N.

1918

on

. cc

. m

g.

Oct

. 26

1,

900

170

1,02

0 15

97.1

“

27

1,90

0 16

5 1.

019

1,58

5.Q

“

28

1,90

0 16

0 1.

020

1,52

9.Q

“

29

1,91

0 16

5 1.

020

1,67

0.O

(‘

30

1,92

0 15

0 1.

020

1,52

9.Q

“

31

1,93

0 14

0 1.

021

1,60

2.7

Nov

. 1

1,94

0 15

5 1.

020

1,55

7.Q

<‘

2

1,93

0 16

0 1.

018

1,60

2.7

‘I 3

1,93

0 16

5 1.

018

1,60

8.4

“ 4

1,93

0 15

0 1.

020

1,60

8.4

TABL

E I.

- I - _

-

-

Urea

N.

Am

mon

ia N.

4m

ino-a

cid

N.

Km

cent

tota

l N

88.4

7

88.9

4 88

.76

89.8

3

85.6

0 84

.52

88.7

7 87

.68

89.4

5

87.7

0

-

mg.

P 1

1.2

1.7

1.5

1.7

1.7

1.6

1.3

1.3

1.5

1.7

w

ceni

!ota

l N

0.08

0.11

0.

10

0.10

0.

11

0.10

0.

08

0.08

0.09

0.

11

m7.

12.8

12.9

11

.6

14.1

31.0

55

.4

14.9

11

.7

12.1

17

.1

,er

cm

‘otaz

N

0.80

0.81

0.

76

0.84

2.03

3.

46

0.96

0.

73

0.75

1.06

mg.

1,41

3.3

1,41

0.5

1,35

8.O

1,

500.

2

1,30

9.6

1,35

4.6

1,38

2-Q

1,

405.

3

1,43

8.7

1,41

0.5

0.5

gm.

phen

ylac

etic

ac

id

in t

he d

iet.

1.0

“ “

“ “

“ “

TABL

E II.

-

‘Olllr

n~

of

urine

.

- Bo

dy

weig

ht.

Spec

ific

grav

ity.

Tota

l N.

- - Ur

ea

N.

Amm

onia

N.

Amino

-acid

N.

- 19

18

9712

. cc

.

Nov

. 9

2,42

0 20

0 “

10

2,42

0 20

0 “

11

2,44

0 24

0 “

12

2,44

0 19

5 “

13

2,42

0 14

5

1.01

8 1.

020

1.01

6 1.

019

1.03

1

m7.

l,Q61

.4

2,09

5.Q

2,

084.

7 2,

020.

2

2,86

9.3

ml.

1,72

9.4

1 8’

5.9

1:&.

5

1,79

4.4

2,51

6.7

per

cent

tota

l N

w.

per

ten

tota

l N

88.1

7 2.

2 0.

11

88.5

5 2.

4 0.

12

88.6

7 3.

1 0.

15

88.8

7 4.

5 0.

22

87.7

1 16

.3

0.57

w.

23.2

23

.8

23.4

22

.6

74.9

?Bt c

ent

totd

N

1.18

1.

14

1.12

1.

12

2.61

2,38

0 22

5 1.

021

3,20

5.5

2,83

1.O

85

.03

10.2

0.

32

81.0

2.

52

Date.

“ 14

“ 15

1.2

gm.

phen

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ac

id

thro

ugh

stom

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tube

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of

this

da

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ratio

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1.

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-

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pm.

cc.

2,83

0 25

5 1.

020

1 2,

720

75

1.03

5 2

2,65

0 48

1.

045

3 2,

580

45

1.04

2 4

2,51

0 47

1.

048

5 2,

420

87

1.04

3 6

2,32

0 70

1.

036

7 2,

220

63

1.04

1 8

2,15

0 98

1.

041

TABL

E III

.

S5

Cc.

D

istil

led

Wat

er

thro

ugh

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mac

h Tu

be

Each

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ay.

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Ur

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1 hin

o-ac

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. -

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pw

cent

!&al

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92.7

8 89

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88.1

5 87

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82.5

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alin

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3,16

6.3

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1,78

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597.

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1,35

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513.

l

6.7

4.5

4.3

3.4

5.6

w

een

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N

0.21

0.

25

0.27

0.

25

0.37

T?.

2,93

5:4

1,59

7.l

1,40

7.g

1,17

7.l

1,24

9.7

26.5

20

.1

18.4

11

.0

91.5

w ee

lz,

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N

0.84

1.

13

1.15

0.

81

6.44

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589.

O

2,18

7.S

84

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9.0

0.35

66

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2.56

I‘

lJ39

4.2

1,66

5.2

87.9

1 4.

8 0.

22

34.5

1.

82

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O

1,79

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88.8

7 6.

7 0.

33

23.0

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15

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947.

7 89

.76

16.8

0.

51

27.0

0.

82

-

t -

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arks

.

Last

da

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fee

ding

. 5 x ,‘: w.

1.0

gm.

phen

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Cellular Metabolism

followed by a decrease. The outputs of urea, ammonia, and ami- no-acids showed increases; the urea absolutely and the others both relatively and absolutely.

Experiment 4.-The rabbit, weighing 3,130 gm., was fed tofukara until the beginning of the experiment, after which it received neither food nor

water. On both the 4th and 5th fasting days, it received 0.5 gm. of phenyl-

acetic acid, twice daily (10 a.m. and 5 p.m.). The acid, neutralized with 10 cc. of sodium carbonate solution, was injected subcutaneously.

The results agree with those obtained in the preceding experi- ments in which the animal received the substance per OS.

Experiments with Phenylpropionic Acid.

Experiment 5.-A rabbit, weighing 2,550 gm., was maintained in N bal- ance. It was given phenylpropionic acid’0 during two periods of 2 days each. The acid was neutralized with sodium carbonate solution and intro- duced through the stomach tube. On both Dec. 15 and 16 the animal

received 1.0 gm. of the acid. On both the 21st and 22nd of Dec., 1.5 gm. were administered.

The total nitrogen and the ammonia excreted were practically unchanged when the acid was introduced. The output of am- monia increased strikingly on both occasions, both absolutely and relatively. In the first case, urea decreased relatively and in the second both absolutely and relatively. The amino-acids were greatly increased.

Experiment 6.-A rabbit, weighing 2,560 gm., in N balance, was treated in the same manner as the preceding one but with larger doses. On Dec.

23 and 24, it received 2.5 gm. daily of neutralized acid and on the 28th and 29th, 3.5 gm. daily.

On the first day in which the acid was administered, the total nitrogen excreted was increased. On the second day, it was nor- mal. There was, also, an absolute increase in the output of urea on the first day. Relatively, however, it was decreased on both days. Both absolutely and relatively the ammoniaand amino-acid excretions were increased on both days on which acid was given. During the second period of feeding the acids, similar changes were observed but of greater degree.

lo The acid was prepared by the reduction of cinnamic acid with sodium

amalgam as described by Erlenmeyer and Alexejeff (Erlenmeyer and Alexe- jeff, Ann. Chem., 1862, cxxi, 375.

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TABL

E IV

.

Day

0 fa

sting

- f $.

- -

? 5 e

_-

Body

“b

”f”’

weig

ht.

urine

--

pm

cc.

3,13

0 25

5 3,

040

60

2,95

0 48

2,

880

51

2,80

0 69

3pec

ific

grav

ity

1.01

3 1.

035

1.04

2 1.

045

1.04

6

2,69

0 85

1.

047

2,58

0 76

1.

040

2,50

0 60

1.

042

2,41

0 64

1.

047

2,33

0 60

1.

047

Reac

tion.

Tota

l N.

Ur

ea

N.

Alka

line

Acid

. “ “ “ “ “ ‘I “ ‘I

-

- Amm

onia

N.

mg.

mo.

;o

z-T’

91

.59

86.4

1 87

.03

89.9

8 84

.93

w.

2,44

3.3

2,23

7.7

1,59

1.5

1,37

5.2

1,52

9.g

1,33

1.4

1,81

5.7

1,63

3.g

2,44

3.3

2,07

5.O

3.9

3.4

2.4

2.6

4.1

Per

een

tota

l N

0.16

0.21

0.

16

0.14

0.17

2,99

2.5

2,56

5.g

85.7

4 6.

3 0.

21

2,32

5.7

2,01

4.O

86

.55

3.4

0.15

1,97

2.6

1,76

8.l

89.6

3 2.

8 0.

14

2,40

0.g

2,14

8.o

89.0

5 3.

9 0.

16

2,49

9.4

2,26

4.6

90.6

6 5.

0 0.

20

-

- !

1 4mino

-acid

N.

-

- W

T.

32.5

29.8

28

.2

33.6

85.6

er

ten

old

N

1.33

1.87

1.

91

1.85

3.50

10.5

3.

65

38.5

1.

66

37.9

1.

92

32.5

1.

35

36.8

1.

48

Rem

arks

.

Last

da

y of

fe

edin

g.

Twic

e (1

0 a.

m.

and

5 p.

m.)

0.5

x c:

gm.

phen

ylace

tic

acid

, di

s-

I-’

solve

d 10

cc

. so

da

solu

tion,

E

inje

cted

hy

pode

rmica

lly.

P “

I‘ ‘I

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TABL

E V.

Date.

lOi8

Dec.

11

“

12

“ 13

“ 14

“

15

“ 16

(‘ 17

“

18

“ 19

“

20

“ 21

“ 22

“

23

“ 24

Body

we

ight

.

2,55

0 2,

560

2,56

0 2,

560

2,57

0

2,57

0

2,57

0 2,

570

2,56

0

2,56

0 2,

570

2,56

0 2,

560

2,57

0

V 1 -- -

‘ohm

u of

m

ine.

-I-

cc.

w.

ml.

t pe

r ce

nm

tota

l N

90.2

8 89

.74

89.1

1 90

.46

86.5

9

:t T

210

1.01

8 1,

989.

4 1,

796.

l 22

5 1.

017

1,95

0.2

1,75

0.o

205

1.01

9 1,

894.

2 1,

687.

g 22

0 1.

018

2,05

1.l

1,85

5.5

210

1.01

9 2,

017.

4 1,

767.

O

2.8

4.1

4.5

5.0

3.9

oer

een

tota

l A

0.14

0.21

0.

24

0.24

0.19

14.9

16.6

17

.7

15.3

69.9

Mr

em

tota

l N

0.75

0.85

0.

93

0.75

3.47

205

1.01

8 2,

028.

6 1,

775.

7 87

.53

4.5

0.22

75

.2

3.71

21

0 1.

018

2,03

4.3

1,83

3.0

90.1

1 4.

3 0.

21

15.3

0.

75

220

1.01

7 1,

950.

2 1,

?41.

2 89

.29

5.3

0.27

14

.9

0.76

21

0 1.

017

1,93

9.o

1,74

5.0

89.9

1 5.

0 0.

26

15.3

0.

79

220

1.01

6 1,

939.

0 1,

765.

3 91

.04

5.6

0.29

14

.7

0.76

21

0 1.

019

2,02

8.6

1,77

1.7

87.3

4 4.

8 0.

24

52.4

4.

06

220

1.01

8 1,

883.

g 1,

627.

7 86

.41

3.1

0.17

84

.7

4.50

24

0 1.

016

1,92

7.g

1,74

6.3

90.5

8 2.

2 0.

11

14.1

0.

73

240

1.01

5 1,

944.

6 1,

752.

l 90

.10

2.0

0.10

13

.5

0.69

3pec

ific

:rsvit

y To

tal

N.

Urea

N.

c

- I -

Amm

onia

N.

- I Amino

-acid

N.

Re

mar

ks.

F 1.

0 gm

. ph

enylp

ropi

onic

acid

F

thro

ugh

stom

ach

tube

. “

“ “

2 %

0

1.5

gm.

phen

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pion

ic ac

id

thro

ugh

stom

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tube

. I‘

“ “

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Date.

Bo

dy

weig

ht,

1918

Dec.

20

“

21

“ 22

“

23

2,56

0 2,

560

2,55

0 2,

560

cc.

240

220

240

220

“ 24

2,

550

230

“ 25

2,

550

235

“ 26

2,

550

235

‘( 27

2,

550

240

“ 28

2,

550

190

“ 29

2,

520

225

“ 30

2,

520

210

“ 31

2,

530

245

Jpec

ific

:ravit

y To

tal

N.

- I Ur

ea

N.

__

mg.

w.

1.01

5 2,

140.

7 1,

942.

z 1.

017

2,00

6.Z

1,79

6.7

1.01

5 2,

174.

4 1,

958.

l 1.

019

2,49

9.4

2,09

1.7

1.02

0 2,

079.

l 1,

715.

4 1.

017

2,04

5.5

1,79

6.l

1.01

6 2,

017.

4 1,

807.

7 1.

016

2,17

9.6

1,95

2.g

1.02

8 2,

348.

l 1,

961.

5

1.02

5 2,

678.

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Y. Hijikata 153

Experiment 7.-The rabbit, weighing 3,120 gm., was fed with tofukara for a time and then fasted. During the experiment it received 35 cc. of water daily. On both the 5th and 6th days, 3.0 gm. of the acid, in 35 cc. of sodium carbonate solution, were introduced through the stomach tube.

The total excretion of nitrogen decreased regularly until the acid was administered. It then increased, with the exception of the seventh day, until death. The urea, also, rose absolutely beginning with the fifth day, but on the 2 days in which the acid was given it decreased relatively. The ammonia excretion paral- leled that of the total nitrogen. There was a great increase in the quantity of amino-acids both absolutely and relatively.

Experiment 8.-A fasting rabbit, weighing 2,610 gm., received, on the 4th and 5th days, two subcutaneous injections daily of 1.0 gm. each of phenylpropionic acid in 20 cc. of sodium carbonate solution.

On both days in which the acid was given, the total nitrogen, as well as the urea and amino-acids, was increased. Relatively, however, the urea decreased. On the second day, the absolute quantity of ammonia was increased but there was relatively no change. The increase in the amino-acid excretion was both abso- lute and relative.

Experiment 9.-The rabbit weighed 3,210 gm. On the 4th and 5th days of fasting, it received, twice daily, 1.2 gm. of phenylpropionic acid. The acid, neutralized with 15 cc. of sodium carbonate solution, was injected subcutaneously.

The result of the experiment was to confirm the foregoing ones.

DISCUSSION.

The experiments reported show that both substances under investigation have the same effect on the distribution of nitrogen in the urine. The effect of the phenylpropionic acid is perhaps smaller.

The total nitrogen, as well as the ammonia, shows no essential change with a small dose. An insignificant decrease is noted in the output of urea. No simultaneous decreases in the excretion of total nitrogen and urea, such as those reported by Desgrez and Guende, in the case of phenylpropionic acid, were observed. The amino-acids in the urine showed a very considerable increase, not

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Cellular Metabolism

only absolutely but relatively. Although no attempt was made to isolate glycocoll, it seems quite probable that the increase may have been largely due to this substance in the form of a conjugate.

The fact that the increase in the amino-acids is accompanied by a decrease in urea suggests the possibility that the amino-acids may arise from urea or its antecedent. The results may be of importance in clarifying our conception of the formation of glyco- co11 in the animal body, no experimental reduction of the urea in the urine having been observed heretofore, on introducing ben- zoic acid or analogous substances.

When the dose was increased the total nitrogen excretion was, without exception considerably augmented, thus confirming the observations of Salkowski on phenylacetic acid. The increases in the absolute and relative quantities of ammonia and amino-acids are remarkably great. The urea also rises but not in proportion to the total nitrogen. The increase in the ammonia, even though the acids were neutralized, favors the view that the simultaneous increases in the quantities of total nitrogen and urea excreted may have been due to a pathological decomposition of tissue proteins. These changes were observed not only with rabbits maintained in N balance but with fasting animals.

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Yoshizumi HijikataURINE

DISTRIBUTION OF NITROGEN IN THE PHENYLPROPIONIC ACIDS ON THE

OF PHENYLACETIC AND METABOLISM: II. ON THE INFLUENCE

PRODUCTS ON CELLULAR THE INFLUENCE OF PUTREFACTION

1922, 51:141-154.J. Biol. Chem. 

  http://www.jbc.org/content/51/1/141.citation

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