VITAMIN C STUDIES IN THE RAT AND GUINEA PIG*

BY JOSEPH L. SVIRBELY

(From the Department of Science, Margaret Morrison Carnegie College, Carnegie Institute of Technology, Pittsburgh)

(Received for publication, September 2, 1936)

The important role played by the small intestine of the rat in the metabolism of incomplete diets as related to the vitamin C content of the liver and gut has been adequately discussed (l).’ More definite evidence with respect to various diets and fatty liver degeneration induced by carbon tetrachloride inhalation indicated that the gut was the probable site of synthesis of ascorbic acid (2). .A more detailed study of this problem was undertaken with the feeding of substances, inorganic as well as organic, which interfered with the normal metabolic activities of the liver and gut.

In addition to the above study with rats, the relationship between cortin and vitamin C was investigated in view of the reported beneficial results of a high sodium, low potassium diet. in the case of adrenalectomized dogs (3). With such a salt diet, adrenalectomized dogs can be maintained in normal condition without cortin for a long period of time. Experimental details from several laboratories indicated that cortin was without any influence in delaying the onset of scurvy in guinea pigs. It was hoped that with a high sodium salt diet further information might be obtained to clarify the connection between cortin’and vitamin C, since guinea pigs dying with severe scurvy show many symp- toms at necropsy which are characteristic of dogs succumbing to adrenal deficiency.

* Much of the material upon which this paper is based was presented before the meeting of the American Chemical Society at Pittsburgh, September, 1936.

‘A recent communication by Zilva (Zilva, S. S., Biochem. J., 30, 857 (1936)) criticizes to a certain extent the interpretation of the data presented in this publication.

543

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544 Vitamin C

EXPERIMENTAL

Young white rats, selected from the various litters, were placed at weaning age on the basal ration of Dog Chow (Ralston Purina Dog Chow). At the beginning of the test period various sub- stances were intimately mixed with ground Dog Chow and fed to the rats. Fresh food was given daily (an intake of 10 gm. of diet per rat). In order to prevent loss by spilling, the diet was mois- tened with water. The weight of the substances given to the animals varied within fairly wide limits but, in general, it was the maximum amount that could be tolerated by the rat as noted by the consumption of the diet. Water was kept before the animals at all times and weekly body weights were recorded.

At the end of the test period the rats were killed by stunning, 18 to 20 hours after the last feeding. The weighing of the organs, extraction with 4 per cent trichloroacetic acid, titration with standardized 2,6-dibromophenol indophenol blue, and estimation of the ascorbic acid content of the organs were carried out as previously described (2).

The experimental details as regards scurvy in the guinea pig will be discussed in a later section.

E$ect of Metallic Xalts-The influence of metallic salts on the body organism has been adequately discussed and an extensive bibliography has been compiled (4, 5), so that no attempt will be made to list the numerous references to the literature on this subject. In the present study the dosage fed, as well as the duration of the test period, was sufficient to insure the full physio- logical response of the rats to the meballic salts. There was no difficulty on refusal of the rats to eat the diet, but a retardation in growth (body weight) was noted in many instances.

The data in Table I indicate that the vitamin C content of the liver and gut was not decreased markedly by any of the salts fed. Occasionally, the general tendency, which had been previously noted with various diets (1,2), of the vitamin C content of the gut to be higher than that of the liver, was not observed. Such devia- tions from the general trend are not of any great significance if the possible catalytic oxidation of vitamin C by traces of metallic salts passing through the intestine is taken into consideration.

The absorption of inorganic salts is quite low, but sufficient

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J. L. Svirbely

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546 Vitamin C

amounts may be stored in the tissues owing to a cumulative storage from large doses. The relation of ascorbic acid content of the organs to the amount of CuSOd fed to the rats is of biologic importance. In vitro experiments have indicated that aqueous extracts of liver inhibit the oxidation of ascorbic acid in distilled water even when Cu or Fe salts have been added (6). The doses of CuSO4 given in vivo, as indicated in Table I, suggest that the body has a protective mechanism whereby the amount of vitamin C is maintained at a fairly high level in the organs. There is some indication that cysteine, cystine, and glutathione play a part in this protective mechanism (7).

The ability of the gut to increase in weight with the feeding of some salts and to approach or even exGeed the weight of the liver presents many interesting problems in physiology. Ordinarily, in young immature rats the gut exceeds the liver in weight, but the reverse weight relationship is found in older rats and the vitamin C content of the gut and liver tends to decrease as the age of the rat increases (2). Injury of the intestinal epithelium by poisoning with metallic salts as sodium fluoride, copper sulfate, or cadmium sulfate may account for the marked increase in weight of the gut. The surprisingly normal values for vitamin C content of the liver and gut of rats fed certain salts, if compared with the relative changes in weight of these organs, indicate that the gut plays a prominent r81e in the synthesis of the antiscorbutic vitamin.

Appreciable amounts of ascorbic acid were present in the organs of rats poisoned with subcutaneous injections of uranyl nitrate (1 mg. daily for 5 days). Refusal of the animals to eat the diet satisfactorily during the last 3 days may account for the slight decrease in ascorbic acid content of the liver and gut. Injections of large doses of thallium or uranyl nitrate in the brief period of 4 hours decreased the vitamin C content of the adrenals only. The marked decrease in ascorbic acid content of the adrenals conforms with previously reported work which showed that thallium salts change the histologic structure of the adrenal cortex (8).

E$ect of Organic Substances-Foreign organic substances intro- duced into the animal body by way of the gastrointestinal tract, injection, or other means are not usually considered in the nature of foodstuffs but as toxic substances. Such toxic substances may

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J. L. Svirbely 547

either be converted into more active pathogenic substances (1,2- benzanthracene, producing cancer under certain conditions in some species of animals) or be detoxicated by conversion into some harmless compound which is eliminated for the greater part from the body through the urine. The gut as well as the liver is normally endowed with important detoxicating powers.

The results of feeding of organic substances at levels indicated in Table II conform in general with previous data which indicate that the concentration of ascorbic acid in the gut tends to be higher than that of the liver. The actual amounts of vitamin C in the organs, the relative increase in weight of the liver and gut (such an increase in weight may be taken as a measure of the detoxi- eating power of that organ), and the retardation in body weight depended to a large extent on the substances fed. It has been shown in other laboratories that many of these substances interfere in one way or another with the metabolic processes of the liver and gut.

Our present knowledge of the metabolism of aromatic com- pounds with the exception of benzene and naphthalene is very scanty. Naphthalenemercapturic acid and phenanthreneglycu- ronic acid have been isolated from the urine of rabbits fed the corresponding hydrocarbons respectively. Dihydroxydihydro- anthracene (which may possibly be formed in the gut by intestinal bacteria), a glycuronic acid, as well as a mercapturic acid deriva- tive of anthracene, has been isolated from the urine of anthracene- fed rats (9). The formation of such compounds as dihydroxydi- hydroanthracene (which rapidly reduces KMn04 in acetone) among other possible derivatives may account for the higher reducing capacity (which can scarcely be attributed to vitamin C) of the liver and gut of rats fed anthracene. Such an explanation may possibly hold true in the case of the phenanthrene-fed rats which also show a higher reducing capacity in the gut than could be normally expected and especially since the feeding of other ring compounds as picric acid, naphthalene, or P-naphthol showed no such increase in the reducing capacity in these two organs. These results indicate that considerable caution should be used in the interpretation of data obtained by dye methods. Many sub- stances, under certain conditions, may reduce the indophenol indicator without being antiscorbutic in nature. The organic

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TABL

E II

Effe

ct

of

Orga

nic

Subs

tanc

es

on

Asco

rbic

Acid

Co

nten

t of

Org

ans

Subs

tanc

es

intim

ately

m

ixed

with

Do

g Ch

ow

fed

daily

200

mg.

* ch

oles

tero

l ...

......

.. 15

“

picr

ic ac

id

......

.....

Cont

rols

......

......

......

....

100

mg.

na

phth

alene

...

.....

400

“ an

thra

cene

...

......

. 10

0 “

phen

anth

rene

t ...

...

20

‘I ox

alic

acid

...

......

. Co

ntro

ls.

......

......

......

. 0.

10

cc.

brom

oben

zene

...

....

0.10

“

“ ...

......

0.

05

“ nit

robe

nzen

e ...

......

. 0.

30

‘* be

nzen

e ...

......

......

23

m

g.

iodo

acet

ic ac

id$.

...

....

Cont

rols

......

......

......

....

100

mg.

fl-

naph

thol.

. ...

......

. Co

ntro

ls ...

......

......

......

.

4 F.

4

(‘ 3

(‘

4 (‘

4 (‘

4 “

4 (‘

4 I*

5 I‘

4 M.

4

(‘ 3

“ 4

“ 3

“

4 “

2 ‘[

,m.

!p.

days

94

151

21

91

105

22

85

141

22

Zays

58

58

59

95

112

17

60

99

130

17

60

99

107

17

60

96

143

17

60

95

141

17

60

75

83

36

72

118

145

22

65

117

121

22

65

120

185

22

65

106

158

22

65

117

215

22

65

115

158

14

123

203

14

55

55

-

mg.

m

g.

Per

Per

orga

n or

gan

I.095

1.

12E

1.05

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73.8

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0.74

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760.

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340.

140.

210.

155.

284.

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80.8

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53.5

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66.7

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2.56

0.13

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5.64

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0.03

2.38

0.07

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5.66

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I.067

0.

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00.0

63.8

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18.6

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72

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2.46

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Vita

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cont

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of r

elativ

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ver

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m 4

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0 gm

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4.85

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02

4.76

5.03

5.

68

7.24

4.

30

4.62

6.

83

6.00

5.

13

4.00

4.

34

4.00

5.18

4.

60

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gm.

final

bo

dy

weig

ht

3.21

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3.51

0

3.62

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3.58

3.

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3.40

5.

19

3.08

3.84

3.

29

* Th

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J. L. Svirbely

substances fed to the rats did not reduce the dye (the substances being dissolved in alcohol), but it is possible that intermediate compounds formed in the body from such substances may have the property of reducing the indophenol indicator.

The addition of iodoacetic acid to the diet yielded data which are of significance in relation to previously reported findings on the function of the intestinal mucosa in the absorption of carbo- hydrates and fats. The synthesis of phospholipids is checked by both phlorhizin and iodoacetic acid, but the absorption of fats is only slightly diminished by the former agent and completely inhibited by the latter substance (10). The phosphorylation mechanism of glucose, fructose, and galactose in the intestinal mucosa, as well as the rate of absorption of these sugars, is inter- fered with by iodoacetic acid (11). Halogenacetic acids have been shown to react with sulfhydryl compounds, chiefly gluta- thione and cysteine, and this action provided an adequate explana- tion for the destruction of glyoxalase activity by such acids (12). Ascorbic acid has been shown to reduce the inhibitory action of iodoacetic acid on the production of acetoacetic acid from butyric or crotonic acid on liver slices (13).

A marked difference exists in the response of the rat to phlor- hizin and iodoacetic acid-the vitamin C concentration in the liver and gut being unaffected by phlorhizin (2), and decreased, as indicated in Table II, by iodoacetic acid. In vitro experiments indicated that iodoacetic acid did not reduce the amount of dye required for titration of ascorbic acid (0.05 mg. of ascorbic acid plus 8 mg. of iodoacetic acid required the same amount of indi’cator as ascorbic acid alone). Incubation experiments have likewise indicated that the protective power of liver in relation to ascorbic acid oxidation was unaffected by iodoacetic acid (7). Conse- quently, the feeding of iodoacetic acid interferes in some way with the synthesis of vitamin C in the rat-probably by the inhibition of the functions of the intestinal mucosa as regards the metabolism and absorption of foodstuffs.

The feeding of the benzene derivatives also yielded data which are of interest as regards the synthesis of vitamin C in the rat. Bromobenzene conformed with iodoacetic acid in the decrease of ascorbic acid in the liver and gut; nitrobenzene showed a decrease in the liver only while benzene was without any apparent effect.

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Vitamin C

The significant amounts of vitamin C in the organs of rats fed nitrobenzene are of interest if the physiological action of this substance is taken into consideration-methemoglobin formation in the blood and the markedly enlarged spleen. Considerable difficulty was encountered in the determination of a satisfactory end-point of the spleen extracts of the nitrobenzene-fed rats so that the values for the vitamin C content in that organ are higher than that actually present.

Bromobenzene leads to fatty degeneration of the liver but the parallel decrease of ascorbic acid in the liver and gut of the rats fed this substance as well as those fed iodoacetic acid suggests that there is a direct interference (the exact mechanism of which is unknown at present) with the synthesis of vitamin C. The decrease in ascorbic acid capacity of the liver and gut by such halogenated compounds can scarcely be attributed to vitamin C playing a major role in the detoxication of these substances. Such a decrease can be more readily explained if it is assumed that the gut is the probable site of synthesis of vitamin C which is then absorbed and utilized in the liver. Any interference with the functions of the intestinal mucosa by such halogenated com- pounds then would lead to a decrease in the vitamin C content in both of these organs.

Relationship of High Sodium Salt to Scurvy-The details as to selection of animals, diet, and scoring of scorbutic symptoms, described in the Sherman-Smith monograph (14), were followed. The animals were divided into two groups, one which received the usual basal diet plus tap water to drink, and the other which received the high salt diet (55 gm. of a 2: 1 mixture of NaCl and sodium citrate per kilo of diet) plus a 0.9 per cent solution of these salts to drink. No difficulty was encountered in the refusal of the guinea pigs to consume the salt ration. On this salt diet about 4 to 5 times as much total salt is consumed as with the usual basal diet. The amount of salt mixture consumed was on a higher level than that fed to adrenalectomized dogs (approximately 1 gm. per kilo (3) in comparison to 2 to 3 gm. per kilo for the guinea pigs). To maintain the adrenalectomized dogs in normal condition with- out cortin, sodium citrate or sodium bicarbonate must be given in conjunction with sodium chloride. At the end of the test period the guinea pigs were killed by stunning, scored for scorbutic

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TABL

E III

Com

paris

on

of

Diet

s in

Sc

urvl~

Avera

ge

weigh

t

- I -2

6 gm

.

219

19

260

22

462

9 62

0 Tr

ace

622

0 25

6 18

28

0 21

52

3 8

625

Trac

e 61

5 0

- -

- I - -_ L It -

Vitam

in C

conte

nt*

Diet

3 .t:

9

-

mg.

Pe

r or

gan

0.01

4 0.

01s

0.02

E

0.01

3 0.

014

0.02

1

w?.

Pm

orga

n

0.3:

0.

3;

0.4(

0.31

0.

36

0.3

-

mg.

Pe

r m

g.

Per

9m.

0.01

0

.Ol

0.02

0.01

0.

02

0.01

w.

Per

00

gm.

final body

m

ight

177

188 94

67

82

15

1 14

6 72

77

81

mg.

m

?.

mg.

m

g.

PW

Per

per

Per

wan

w.

977%

. gm

.

1.01

9 0.

03

0.02

0.0

1

1.01

9 0.

05

0.01

0.

0:

1.01

9 0.

05

0.02

0.

0:

I.013

0.

04

0.01

0.

0:

1.01

4 0.

04

0.01

0.

0:

I.019

0.

05

0.01

0.

0:

wag.

0 0 0.25

0.

25

0.50

0 0 0.

25

0.25

0.

50

gm.

17

13

26

26

30

gm.

357

512

440

376

328

362

494

344

355

312

Sher

man

-Sm

ith

basa

l

High

so

dium

sa

l

19

40

73

73

73

23

23

72

72

72

0.15

0.

22

0.30

0.21

0.

20

0.27

IC

rc

IC a 0 0 -

* Gu

inea

pigs

kil

led

by

stunn

ing.

t Ha

d be

en

fed

0.5

mg.

of

vit

amin

C for

39

day

s pr

eviou

sly.

-..

Y._

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552 Vitamin C

symptoms, the organs extracted with trichloroacetic acid, titrated with standardized 2,6-dibromophenol indophenol blue, and esti- mated for vit,amin C content.

The data in Table III indicate that there was no significant difference in survival period or scorbutic symptoms of the guinea pigs on the basal diets alone. Subnormal (0.25 mg.) as well as normal (0.5 mg.) doses of ascorbic acid likewise showed no marked deviations in response of the guinea pigs on the diets. A slight individual weight variation was apparent, but this weight varia- tion is often encountered in vitamin C assay work and may be more pronounced as regards sexes of the experimental animals. The titration values of the organs, survival period, scoring of scorbutic symptoms, etc., conform with previously reported results (15), that in the guinea pig vitamin C is the essential factor in preventing scurvy and that cortin is without any effect in delaying the onset of scurvy. Adrenalectomized dogs can be maintained in normal condition without cortin on a high sodium salt diet because they synthesize their own vitamin C. Guinea pigs dying of scurvy show a much more marked hypertrophy of the adrenals (based for uniformity on weight of adrenals in mg. to 100 gm. of final body weight) than those which had received subnormal or normal doses of ascorbic acid.

SUMMARY

1. The feeding of metallic salts, some of which poison the intesti- nal epithelium, does not prevent the synthesis of vitamin C in the rat. The body has a protective mechanism which prevents the catalytic oxidation of vitamin C with CuSO4.

2. Halogenated compounds, as bromobenzene or iodoacetic acid, interfere with the synthesis of vitamin C in the rat. Other organic substances fed did not show this property.

3. Guinea pigs on a high sodium salt diet (a mixture of sodium chloride and sodium citrate added to the usual basal diet) succumb in the usual time interval and with the same severity of scorbutic symptoms as on the Sherman-Smith basal diet. Vitamin C is the limiting factor in preventing scurvy and, in those animals which are susceptible to this deficiency disease, is necessary for the utiliza- tion of the cortical hormone or the salt mechanism apparently controlled by cortin.

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BIBLIOGRAPHY

1. Hopkins, F. G., Slater, B. R., and Millikan, G. A., Biochem. J., 29, 2803 (1935).

2. Svirbely, J. L., Am. J. Physiol., 116, 446 (1936). 3. Allers, W. D., Nilson, H. W., and Kendall, E. C., Proc. StuflMeetings

Mayo Clin., 11, 283 (1936). 4. Hart, E. B., and Elvehjem, C. A., in Luck, J. M., Annual review of

biochemistry, Stanford University, 6, 271 (1936). 5. Sollmann, T., Pharmacology, Philadelphia and London, 4th edition

(1932). 6. Kellie, A. E., and Zilva, S. S., Biochem. J., 29,1028 (1935). 7. Mawson, C. A., Biochem. J., 29, 569 (1935). 8. Hecke, F., Virchows Arch. path. And., 269,28 (1928). 9. Boyland, E., and Levy, A. A., Biochem. J., 29,2679 (1935).

10. Terroine, E. F., in Luck, J. M., Annual review of biochemistry, Stan- ford University, 6, 234 (1936).

11. Chaikoff, I. L., in Luck, J. M., Annual review of biochemistry, Stanford University, 6, 205 (1936).

12. Dickens, F., Biochem. J., 27, 1141 (1933). 13. Quastel, J. H., and Wheatley, A. H. M., Biochem. J., 28,1014 (1934). 14. Sherman, H. C., and Smith, S. L., The vitamins, American Chemical

Society monograph series, New York, 2nd edition (1931). 15. Svirbely, J. L., J. Biol. Chem., 111,147 (1935).

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Joseph L. SvirbelyGUINEA PIG

VITAMIN C STUDIES IN THE RAT AND

1936, 116:543-553.J. Biol. Chem. 

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