288 A M E R I C A N J O U R N A L OF D I G E S T I V E D I S E A S E S

Andrewes and Herder, our Str. mitis I I ; one with our Str. mitis IV and two with our Str. mitis I. Bargen 's s t ra ins in our hands did not exhibit any apparent dif- ferences f rom our s t rains in (1) the action on blood plates, (2) heat resistance qualities or (3) pathogeni- city in rabbits.

8. There would, therefore, appear to be no reason

to regard the streptococci described as Bargen 's diplo- streptococcus as being essentially different f rom other types of alpha streptococci commonly recovered i rom the human al imentary t rac t and his s t ra ins may be- long to the species Streptococcus faecalis, to Str. mitis to Str. salivarius.

R E F E R E N C E S Andrewes, F. W. and Herder, T. A.: A Study of Streptococci Patho-

genic for Man. Lancet, 2:708, Sept. 15, 1906. Bagger, S. V.: The Enterococcus. Jour. Path. and Bact., 29:225, 1926. Bargen, J . A. : Chronic Ulcerative Colitis. Arch. Int. Med., 45:559,

1930. Besson, A.: Practical Bacteriology, Microbiology and Serum Therapy.

Trans. 5th French Edition by Hutchens, p. 609. Longmans, Green and Co., London, 1913.

Brown, James Howard: The Use of Blood Agar for the Study of Streptococci. Monographs of the Rockefeller Inst. for Med. Res., :No. 9, pp. 1-122, 1919.

nible, J . Henry : The Enterococcus and the Faecal Streptococci: Their Properties and Relations. Jour. Path. (:rid Bact., 24:3, 1912.

Escherich: Darmbakterien des S~iuglings, p. 89, 1886. Gordon, M. H . : A Ready Method of Differentiating Streptococci and

Some Results Already Obtained by its Application. Lancet, 2:1400, 1905.

Hirsch and Libman : Ein Pall von Streptokokken-Enteritis in S~ugling- salter. Centratb. f. Bakt. u. Parasitenk., 23:369, 376, 1897.

Holman, W. L. : Classification of Streptococci. Jour. Med. Res., 34:377, 1916.

Houston, A. C.: Report to M.O.H. to Local Government Board, 33:472, 528, 1903-4.

Mac~: Trait~ pratique de Bact6riologie. Paris. Bailli~re et Fils. 1912.

Orcutt, M. L. : A Study of Enterococci f rom the Digestive Trac t of Calves. Jour. Bact., 11:129, 1926.

Rafsky, Henry A. and Manheims, Perry S. : The Significance ~f the Bargen Organism as an Etiologic Factor in Ulcerative Colitis. Am, Jour. Med. Sci.. 183:252, 1932.

Rankin, F. W., Bargen, J. A. and Buie, L. A.: The Colon, R~ctnm and Anus. W. B. Saunders Co., Philadelphia, 1932.

Thiercelin: Sur un Diplocoque Saprophite de l 'Intestin Susceptibile de devenir Pathog~ne. Compt. rend Soc. Biol., 51:2(;9, 551, 1899.

Thomson, David and Thomson, Robert: Historical Survey of Researches on the Streptococci. Annals of the Pickett-Tho~rson Res. Lab., 3-1- 316, 1927.

Topley, W. W. C. and Wilson, G. S.: The Principles of Bacteriflogy and Immunity. Wm. Wood and Co., New York, 1936.

Torrey, John C. and Montu, Elizabeth: The Cultural and Agglutinative Relationships of Intestinal Streptococci. Jour. Inf , Dis., 55:340, 1934.

Torrey, John C. and Montu, Elizabeth: Comparative Observatiors on Streptococci from Human Gastro-Intestinal Ulcerations and from Bovine Mastitls. Jour. Inf . Dis., 58:105, 1936.

Welch, Henry : Classification of the Streptococci of Human Feces. lout. Bact., 17:413, 1929.

Zinsser, Hans and Bayne-Jones, Stonhope: A Textbook of Bacteri(~logy. D. Appleton-Century Co., New York, 1934.

IV. An Experimental Study of the Hydrogen-Ion Concentration and Chemistry of Bile, its Effect Upon Stones, and a Suggestion as to

Therapeutic Application of Ox-Bile in Gall Bladder Disease By

SAMUEL MORRISON, MAURICE FELDMAN, JOHN C. KRANTZ, JR.

and FRANCES F. BECK

BALTIMORE, MARYLAND

T H E R E are many factors to be considered in the etiology of gall stones. Although much has been

wri t ten regarding the format ion of stones there are but few significant contributions on this subject. I t is noteworthy tha t but little investigative data exist on the hydrogen-ion concentration of bile and many of these have been obtained f rom fistulous bile in both humans and animals. As to the relation between the hydrogen-ion concentration of bile and the formation of gall stones, the l i terature is especially scant.

pH D E T E R M I N A T I O N S IN NORMAL ANIMALS AND MAN

Hydrogen-ion determinations by the electrometric method were made of the gall bladder bile f rom the guinea pig, dog, rabbi t and the human. The pH in a series of 26 normal guinea pigs averaged 8.9. In the normal dog the pH of a series averaged 6.2. The rabbi t gall bladder bile was 8.8; cat 5.3 (Okada) ; ox 7.5 (Okada). The normal human gall bladder bile ranged between 7.0 and 7.5. These findings indicate that the hydrogen-ion concentration of bile varies in different

~From the Research Section of the Depar tment of Gastro-Enterology and the Depar tment of Pharmacology, School of Medicine, Uni- versity of Maryland.

The expense of this work was defrayed in pa r t by the Julius Frieden- wald Experimental Fund.

Submitted November 5, 1937.

species and demonstrates tha t in the herbivorous animal the bile is normally alkaline while in the carni- vorous animal the bile is more likely to be acid. On the other hand, in the human subject, an omnivorous species the hydrogen-ion concentration of gall bladder bile is close to the neutral point.

T H E E F F E C T OF DOG'S BILE ON STONES IN VIVO

In a previous study (1) the authors confirmed the findings of others (2), tha t gall stones of the choles- terol type placed in the dog's gall bladder would dis- solve. Numerous explanations have been advanced to account for this phenomenon, but none seems to be en- t irely satisfactory. A constant finding in the dog's gall bladder bile is its acidity which possibly may be one of the factors concerned with its capacity to dissolve stones. I t occurred to us tha t since in the dog there ~ a s apparently a relationship between the pH of bile a ad the dissolution of gall stones it would be desirable to select an animal with an alkaline gall bladder bile in order to determine its effect upon similar gall stom.~s. The guinea pig was selected for these experiments. W'e were able to demonstrate tha t gall stones placed in tlae guinea pig's gall bladder were not affected by the

MORRISON, F E L D M A N , K R A N T Z AND B E C K - - E X P E R I M E N T A L S T U D Y OF B I L E C H E M I S T R Y 2 8 9

alkaline bile. Fur thermore, it was also noted that no change occurred in the pH of bile owing to the presence of stones.

COMPARATIVE S T U D I E S OF NORMAL BILE AND F I S T U L O U S BILE IN R E L A T I O N TO

T H E I R E F F E C T S UPON G A L L S T O N E S AND HYDROGEN-ION CONCENTRATIONS

As it has been shown tha t human gall stones will dissolve in normal dog's acidic bile and will not dis- solve in the normal alkaline bile of the guinea pig it seemed important to determine what effect a dog's alkaline bile would have on the dissolution of human stones.

In a previous communication (3) we have shown tha t the normal dog's acidic bile could be made alkaline by the production of a fistula which prevents concentra- tion of the bile by the gall bladder. These studies demonstrated tha t when gall stones were implanted into the gall bladder through the fistulous opening, the dissolving effect of the bile was lessened, and in fact in some instances there was actually an increase in weight of the stones. These observations seem to indi- cate tha t alkaline bile may have some tendency to favor the format ion of stones but this has not as yet been definitely proved.

I t is interest ing in this connection to point out tha t Neilson and Meyer (4) observed tha t in gall bladders of various laboratory animals injured by infection the pH of the bile remains constantly identical with that of the secretion derived f rom the liver owing to the inabil i ty of the gall bladder to br ing about its usual change in the bile.

E F F O R T S TO CHANGE T H E pH OF BILE BY F E E D I N G OF VARIOUS SUBSTANCES

(a) . CHEMICALS Gall bladder bile possesses a high buffer capacity

and it is difficult to alter its hydrogen-ion concentra- tion. This applies also to the concentrations of its various constituents. The feeding of various chemical

substances to animals has little tendency to change the hydrogen-ion concentration of gall bladder bile. Even drugs known to be excreted part ial ly by the liver such as salicylic acid showed no influence upon the pH of gall bladder bile. Ottenheimer and Kahn (5) found tha t the oral adminis trat ion of sodium citrate, sodium bicarbonate or hydrochloric acid failed to cause any significant al teration in the pH of bile. They also noted tha t solutions of phenoltetrachlorphthalein and phenoltetraiodophthalein when injected intravenously in animals seemed to be excreted in a colloidal s tate and have no effect on the reaction of bile.

(b). FOODS Since the chemicals employed had little effect upon

the pH of bile it occurred to us tha t feeding experi- ments might change the pH. Three normal dogs were placed upon a vegetable diet consisting of lettuce, carrots, cabbage, etc. Af te r a period of 18 days the bile was obtained directly f rom the gall bladder and hydrogen-ion determinations made. The following pH values were obtained in the three animals--6.92, 6.29 and 6.26--which indicates that the feeding of vege- table diet to dogs had no significant effect on the pI-I of the bile. In another experiment four guinea pigs were fed a meat diet in order to determine whether their alkaline bile couId be made acidic by this change of diet. Af te r a feeding period of 14 days the follow- ing pH determinations were obtained--8.74, 8.63, 8.86, 8.64. These are essentially normal values for the guinea pig, and thus it is readily seen that the change f rom a herbivorous to a carnivorous diet produces no signi- ficant change in the pH of bile in these normal animals.

Bronner (6) in 1933 studied cholecystectomized human subjects with a tube in the ductus choledochus and observed tha t by varying the diet he was able to shif t the pH of the bile to the alkaline side with a vegetable diet and to the acid side with a meat diet. Bronner believes that variat ions in the pH and changes that occur concomitant with it are important factors

TABLE I E]fect of powdered ox-bile U.S.P. on bile pH and stone weight in dogs. Eight grams daily by mouth

Do~

1

4

6

7

8

9

10

I I

12

13

14

Before Implantat ion of Stone

Days p i t Fed

8.04 18

6.44 18

8.70 18

6.23 7

6.55 7

8.46 7

8.24 7

7.5 11

8.02 11

8.18 11 6.32 21

7.10 14

7.10 17

6.65 17

After Implantat ion of Stone

Days pH Fed

8.76 90

8.80 90

8.84 90

8.05 27

7.55 39

7.52 39

None 30

None 67

8.27 67

None 68

6.12 60

None

7.47

6.48

40

42

Total No. Days F e d

108 I

= i 34

46

46

37

78

73

79

31

57

59

Weight of Stone in Grams

Implanted

1.076

3.318

0.998

0.937

1.428

0.504

1.084

1.037

0.914

1.190

0.731

0.893

1.818

Recovered

0.046

0.069

0.879

0.226

0.240

0.112

1.O0O

0.112

None

0.328

None

0.232

0.105

Weight Loss in Stone Grams

1.030

3.249

0.119

0.711

1.188

0.392

0.084 (Abscess G.B.)

0.925

No stone found

0.862

No stone found

Weight Loss in Stone Per Cent

95

98

12

68

94

72

0.611

1.713

7.7

89

74

83

77

2 9 0 A M E R I C A N J O U R N A L OF D I G E S T I V E D I S E A S E S

in the origin of stones. Notwithstanding Bronner's observations on the effect of food upon fistulous bile in the human subject it is our opinion that these changes actually occur in liver bile and not in normal gall

The pH of this bile was determined immediately with a glass electrode. Following ox-bile feedings the oH values approximate those of liver bile. The resul;s are recorded in Table I.

TABLE II Effect o.f ox-bile on bile pH and stone weight in dogs. 0.6 grams daily by "mouth

Before Implantatio:

Dog of Stone

pH

1 6.48

2 None

3 None

4 7.35

5 6.68

6 6.68

After Implantat ion of Stone

pH

None

,%03

6.89

6.61

6.41

6.21

Total Days Fed

42

77

82

82

82

77

Weight of Stone in Grams

Implanted

0.793

1.713

1.952

1.138

Recovered

None

0.092

1.600

0.443

Weight Loss Grams

1.621

0.352 (cale.)

0.695

Weig~ t Loss %

95

18

61

bladder bile. Our experience, at least in the normal dog and guinea pig, does not confirm Bronner's find- ings in the fistulous human subject.

(c). VITAMINS

In a previous communication (1) we reported, as the result of our experiments, that the feeding of vitamins A, B-complex and D had no sigIfificant effect upon the formation or dissolution of gall stones in the dog.

(d). OX-BILE

Since few drugs are known to effect the hydrogen- ion concentration of bile we decided to use powdered Extract Ox-Bile, U. S. P. as an oral medication. Fourteen dogs were utilized in this experiment. Two tablespoonfuls (8 grams) of ox-bile were mixed with the dog's rations and fed over a period of weeks. Inter- esting results were obtained from these experiments. It was found, for instance, by feeding large amounts of ox-bile that the dog's normally acidic bile could be changed to alkaline. No untoward effects developed from this dosage of bile.

Following the oral administration of ox-bile it was observed that the gall bladders were considerably dis- tended. The bile was obtained aseptically by hypo- dermic needle puncture. Its color was altered from the normal dark brown to a striking deep olive green.

T A B L E

Effect of Keto bile acid on bile pH and stone

THE E F F E C T OF F E E D I N G OX-BILE A F T E R IMPLANTATION OF GALL STONES

INTO THE GALL BLADDER In 14 animals which had previously been fed daily

with 8 grams of ox-bile, a cholesterol gall stone was placed in each gall bladder to determine the effect of this alkaline gall bladder bile upon the stones. After a varying period of time (see Table I) it was observed that a marked diminution in the size of the gall stones took place. It seemed therefore that the hydrog(n-ion concentration of the bile was not the sole factor in the dissolution of gall stones as was suggested in our previous investigations (3, 7). However, chemical analysis of these biles resulting from the feeding of ox-bile disclosed a decrease in bile acids and a diminu- tion of protein. The diminished amount of proteiln re- sulting from dilution binding less of the normally occurring cations of bile, may account in part fcr the alkalinity.

THE E F F E C T OF F E E D I N G SMALL AMOUNTS OF OX-BILE

Inasmuch as large doses of ox-bile changed tl~e pH of dog's acidic bile to alkaline it was of interest 1:o de- termine whether a similar response followed small doses, approximating the normal human dosage Six dogs were observed in this experiment. Two, ten grain capsules of ox-bile daily were administered orally to each dog. t t is noteworthy that in these experiments the pH of the dog's gall bladder bile did not change

I I I

weight in dogs. 0.7 grams daily by mouth

Dog

1

2

3

4

5

Before Implantat ion of Stone

pI-I D~ys Fed

7.01 26

6.62 46

6.62 28

7.02 28

6.37 28

After Implantat ion I I of Stone Total

/ Days [ O H Days F e d Fed

6.11 37 ] 65 /

6.17 38 84

7.21 37 65

6.30 37 65

7.10 37 65

Weight of Stone Implanted

1.410

1.244

1.098

1.032

1.352

Weight of Stone Recovered

Weight Loss Grams

0.654

0.782

0.507

0.493

0.746

0.756

0.462

0.591

0.539

0.606

Weight }Loss %

54

36

54

52

45

M O R R I S O N , F E L D M A N , } ( R A N T Z A N D B E C K - - E X P E R I M E N T A L S T U D Y O F B I L E C H E M I S T R Y 29i

following the administration of such small doses. These experiments would indicate that to obtain re- sults in gall bladder disease larger doses would be re- quired in order to influence the composition of bile and thereby probably obtain better therapeutic results (see Table I I ) .

CHEMICAL STUDIES

Chemical studies have been made of the gall bladder bile following the feeding of bile salts. Our results

are summarized in Table IV. It is interesting to note in studying this table that the effect of feeding large

TABLE IV Summary of averages in bile studies

No. of Dogs*

8

19

8

T r e a t m e n t

N o r m a l

Ox-bile**

Ox-bi]e*** A v e r a g e

Keto bile acids ~*** A v e r a g e

p H

6.17

7.68

6.66

6.59

Total Solid G r a m s %

22.77

17.84

21.67

19.19

Muein G r a m s %

1.29

0.75

1.67

0.87

Ash G r a m s %

2 . 0 1

1.72

0.69

1.79

Alka l in i ty %

0.78

0.57

6.37

0.62

L ipo ids G r a m s %

6.51

4.78

6.09

5.16

B i l e Acids %

6.78

5.0

6.59

5.36

*Refe r s to a l a r g e r se r ies t h a n is recorded in the o ther tables. **8 g r a m s daily. ***0.6 g r a m daily. ****0.7 g r a m daily.

THE EFFECT OF FEEDING MIXED KETO BILE ACIDS

Five dogs were observed in this experiment. To each animal 3, 33/~4 grain tablets ~ were administered daily by mouth. The results indicate that on the whole the feeding of keto acids in small dosage did not ma- terially change the pH of bile. It would seem at least from these few experiments, that the dissolution of stones was less than was found in the control or after the feeding of ox-bile. (see Table I I I ) .

T A B L E V

pH of human bile

Case A i l m e n t p i t

1

2

3

4

5

6

7

8

9

I0

11

12

13

14

15

16

17

18

19

Gall S tones

p,

Cholecyst i t is

P a n c r e a t i c Cancer

P a n e r e a t i t i s

8.65

8.04

8.26

8.60

7.44

8.70

8.46

8.64

8.02

8.05

7.90

7.2

7.71

7.55

7.4

7.63

7.31

7.60

7.18

-*This m a t e r i a l w a s k ind ly suppl ied to us by G. D. Sear le & Co., Chicago, I l l inois.

doses of ox-bile is to dilute the vaL'ious constituents of the normal dog's bile. This is a.~soeiated with an in- crease in the pH. On the other hand, small doses cf ox-bile have been found to be ineffective in changing the chemical proportions of the various constituents nor do they change the hydrogen-ion concentration. These experiments suggest that feeding of ox-bile in small doses as is ordinarily administered in the human subject, is therapeutically ineffectual.

Following the feeding of keto bile acids it is of interest to note that although there was considerable dilution of the chemical constituents of the bile the pH was not signifieantiy altered. By comparing the effect of ox-bile with keto bile acids it is readily observed that the ox-bile in large quantity appears to have a greater diluting effect.

HUMAN BILE As the result of our studies on animals, we became

interested in the hydrogen-ion concentration of gall bladder bile in human eases of pathological and normal gall bladders. Biles were obtained at operation from gall stone eases and hydrogen-ion concentrations were determined on the fresh bile. Strikingly enough, it was noted that in each ease of gall stones which we examined, the hydrogen-ion concentration of the bile proved to be alkaline. These studies corroborate the data already obtained in our animal experimen- tation. In 15 human subjects possessing gall stones the hydrogen-ion concentration determinations obtained are higher than normal. (See Table V). We have also found that the type of stone did not significantly in- fluence the pH in these human eases. The results of other pathological conditions are also recorded in this Table.

CONCLUSIONS

1. In a study of gall bladder bile of different species one of the most striking features is the ap- parent immutability of its characteristic hydrogen- ion concentration.

2. The pH of bile does not readily change following various forms of therapy.

3. It is our belief that various foods do not signi- ficantly alter the pH of gall bladder bile.

292 AMERICAN JOURNAL OF DIGESTIVE DISEASES

4. The feeding of large doses of powdered extract ox-bile U. S. P. changes the dog's normal acidic bile to alkaline and also dilutes the chemical constituents. Feeding of smaller doses does not change the pH of gall bladder bile in the dog nor does it have any dilu- t ing effect.

5. The feeding of keto bile acids (0.7 gram per day) did not change the pH of gall bladder bile in the dog nor did it br ing about as grea~ a dissolution of the stones as is noted in the normal and ox-bile fed animal. This dosage of keto bile acids approximated

the human dose. The diluting effect was much less marked than in feeding large doses of ox-bile.

6. The pH of bile is not the sole criterion i~L the dissolution of gall stones, as is demonstrated by the fact that even though large doses of ox-bile chang e the pH of dog's acidic gM1 bladder bile to the alkaline side, they do not significantly influence the degree of dis- solution of implanted human cholesterol stones. Ill our opinion, the dissolution of the stones by the alkaline bile after the feeding of large quantities of ox-bile, is due to its cholagogue effect.

R E F E R E N C E S 1. Morrison, S. and Feldman, M.: Etiology of Gall Stones. 1. The

Solvent Action of Vitamins on Gall Stones. An Experimental Study. Review of Gastro-enterology, 4:120, June, 1937.

2. Walsh, E. L. and Ivy, A. C. : Observations on the Etiology of Gall Stones. Ann. Int. Med., 4:134, 1930.

Walsh, E. L. : Etiology of Gull Stones. Arch. Path., 15:698, 1933. Ivy, A. C.: Observations on the Etiology of Gall Stones. Proc. Internat. Assemb. Inter-State Post Graduate Med. Assn. of N. America, 6:575, 1931.

3. Feldman, M., Morrison, S., Cart , C. J . and Krantz, J. C., J r . : A Contribution to the Etiology of Gall Stones. I I I . A study of the hydrogen-ion concentration of gall bladder bile and its effect upon gall stones. Am. Jour. Dig. Dis. and Nutrit. , IV :223, June, 1937.

4. Neilson, N. M. and Meyer, K. F. : The Reaction and Physiology of

the Hepat ic Duct and Cystic Bile of Various Laboratory Animals. J. Infectious Dis., 28:510, 1921.

5. Ottenheimer, R. and Kuhn, J. : Relative Immutabil i ty of Hydrogen- ion Concentration of the Bile. I ts Buffering Effect in Bacteri- cidal Experiments. Proc. Soc. Exper. Biol. and Med., 29:573, 1931-32.

6. Bronner, H. : Der Einfluss Der Ern~hrung auf die Wassersiaff-ion enkonzentration Der Galle. Klinisehe Blobaehtungen. KZnische Wchnsehr., 12:1562, 1933.

Wasserstaffionenkonzentration der Galle und Steinbildung. Arch. f. Klin. Chit., 180:597, 1934.

7. Feldman, M., Morrison, S. and Krantz, J . C., Jr . : Etiology ,)f Gall Stones. II . The Relationship of the p H of Bile to the Formation and Dissolution of Gall Stones. Am. Jour. Dig. Dis. and Nutrit. , IV :13, March, 1937.

The Effect of Estrogenic Hormone on Gastric Acidity t By

LEON SCHIFF, M.D. and

HENRY FELSON, M.D. With the technical assistance of

JANE GRAFF, B.A. and

BETTY MEYER, M.A.

T HE well known fact that the modal gastric acidity in man is higher than in woman, and conversely

the greater frequency of anacidity in the female (Bloomfield), suggests a possible relationship between sex hormones and gastric secretion. The effect of "Theelin" on gastric acidity has been studied in the dog by Winkelstein, who observed no constant change.

As we were unaware of any similar observations made in man or woman, it was decided to administer Theelin to a group of human subjects. We were par- ticularly interested to see whether or not achlorhydria could be produced.

METHOD USED

A group of twenty individuals was selected, consist- ing of twelve males and eight females (including six castrates). The ages varied between 23 and 75 years with an average age of 42 years. The patients were ambulatory and reported to the laboratory in a fast ing state. They were instructed not to swallow any saliva.

*From the Depar tment of In ternal Medicine, Universi ty of Cincinnati and the Medical Service, Cincinnati General Hospi ta l

~Study made possible by a special g ran t f rom Parke-Davis & Company and through the kind cooperation of Dr. E. A. Sharp.

:~Kindly furnished by Parke-Davis & Co. Submit ted November 23, 1937.

A Rehfuss tube was introduced into the stomach and the fasting contents were withdrawn. One-half rag. of histamine phosphates was given subcutaneously and continuous aspiration carried out in 10 minute pe:?iods for a total of one hour. The presence of bile and mucus was recorded.

Pr ior to the institution of treatment, two or more gastric analyses were done at intervals of 5 to 10 days. During treatment the analyses were continued at intervals of one to two weeks and af ter t reatment at intervals of about two weeks.

The patients were arbi trar i ly divided into three groups. The first group was composed of nine men who were given relatively large doses of "Theelin" over a short period of time. The group was restricted to men because of their tolerance for larger individual (loses of the hormone (Table I) . The doses ranged from 4,000 to 20,000 International Units per injection, the usual dose being 10,000 units. The average duration of treatment was 27 days; the average number of in- jections, 21, and the average duration of study, 9 months (Table 1).

The second group, composed of four women (3 castrates) and three men, received smaller doses of