the elimination of various dyes from the pavlov pouch of dogs

1VIORRISON, REEVES AND G A R D N E R - - T H E ELIMINATION OF VARIOUS DYES FROM THE PAVLOV POUCH OF DOGS 551

good d r a i n a g e depends, not upon the n u m b e r of da i ly evacuat ions , bu t upon the t h o r o u g h n e s s of d ra inage . Many pa t i en t s will r e p o r t f a i l u r e because the bowels have moved bu t once in 24 hours , whi le t hey bel ieve t h a t two o r more evacua t ions a re n e c e s s a r y ; or because the quan t i ty , in t h e i r e s t ima t ion , is not sufficient; or because the stool is not w a t e r y and expel led wi th ex- p los ive force as fo l lowing a ca tha r t i c .

C O N C L U S I O N S

1. The mechan i sm of no rma l bowel d r a i n a g e depends upon the m a i n t e n a n c e of a difference, on the ac id side, be tween the p H and the iso-e lect r ic po in t of the feces.

2. The pH of t he feces, and hence no rma l d ra inage , can quickly be contro l led by the a d m i n i s t r a t i o n of s t a r c h in excess of the p a n c r e a t i c th reshold .

The Elimination of Various Dyes from the Pavlov Pouch of Dogs By

SAMUEL MORRISON, M.D. ~

DAVID L. REEVES, M.D.t

and

RAYMOND E. GARDNER, Sc.D.$ BALTIMORE, MARYLAND

T H E e l imina t ion of dye stuffs by the g a s t r i c mucosa in va r ious pa tho log ica l condi t ions of the s tomach

and the s tudy of th i s e l imina t ion expe r imen ta l l y in an ima l s has received r a t h e r ex tens ive recogn i t ion in recen t years . However , va luable con t r i bu t i ons such as those by Dawson and Ivy, K o b a y a s h i and H e n n i n g a re a t such var iance , t h a t f u r t h e r s tud ies on dye e l imina- t ion were h a r d l y poss ible w i thou t r e - i n v e s t i g a t i n g a t l eas t those po in ts of d i f ference so s t r i k i n g l y obvious in the above repor t s . F u r t h e r m o r e any eva lua t ion of dye e l im ina t i on - s tud i e s makes i t i m p e r a t i v e t h a t t he d i f ferences a l r e ady r e f e r r e d to e i t h e r be verif ied or d i sproved .

Cel lu lar and h i s to-chemica l s tud ies which may ex- p la in the mechan i sm of dye e l imina t ion as well as i ts poss ib le c l in ical app l i ca t ion depend upon end re su l t s w i th which all a u t h o r s a r e in ag reemen t . In the case of dye e l imina t ion the re is def ini te d i s ag reemen t . Hence, we have i n i t i a t ed the p r e s e n t expe r imen t s on dogs w i th Pavlov pouches us ing a l a rge n u m b e r of dyes, which were tes ted and s tud ied a f t e r t h e i r in jec- t ion in a p p r o x i m a t e l y 1% aqueous solut ion, i n t r aven - ously, i n t r a m u s c u l a r l y , and t h r o u g h o the r channels .

D Y E S T U F F S E L I M I N A T E D BY T H E S T O M A C H T H R O U G H P A V L O V P O U C H F O L L O W I N G

I N T R A V E N O U S I N J E C T I O N 1. N E U T R A L R E D ~ A z i n e - - Weakly Bas ic - - ( Ind i -

c a t o r ) - 1 % - 4 0 c.cs.--toxic in tha t quantity. Does not s tain the rams. (mucous membranes) of the stomach or tissues. Through in less than 5 minutes. Also comes through in 1 c.c. quantities used intravenously, and in 1% strength.

2. N E U T R A L VIOLET~Az ine - -Weak ly Basic-- l% - -20 c.cs.--not toxic to the animal in the amount used. Amply through within 5 minutes. Mms. of the stomach stained pinkish-purple.

3. MAGDALA R E D - - A z i n e - - Bas ic - - l % - - 25 c.cs. Mms. stained slightly pink. Not toxic.

"~Assistant Professor, Department of Medicine, Clinic of Gastro-enter- ology, University of Maryland.

tFormerly Assistant in Surgery and Hunterlan Fellow in Surgery, Johns Hopkins University.

SAssociate, Department of Immunology, Johns Hopkins University. Submitted August 22, 1935.

4. S A F R A N I N O--Azine--Basic--1%--25 c.cs. slightly toxic in this amount. Mms. of the stomach stained pink. Comes through definitely, but not to a considerable extent. This is an etherized dog; stomach brought out and examined.

5. METHYLENE BLUE--Thiazine Basic-- l%--20 c.cs. Slightly toxic. Mms. of the stomach stained sl ightly blue. Comes through when 1 c.c. of a 1% solution is injected and not toxic.

6. M E T H Y L E N E BLUE (SPECIAL) - - Thiazine-- Basic-- l%--35 c.cs. Mms. of the stomach not noticeably stained with this quantity. Tissues, tongue, etc., slightly stained. Net toxic. Comes through definitely although not as much as plain methylene blue.

7: M E T H Y L E N E BLUE MERCK--U.S.P.X.--Thia- z ine--Baslc-- l%--30 c.cs. Somewhat toxic. Mms. of stomach, and the tissues not noticeably stained with such an amount. Comes through better than methylene blue, special.

8. NEW M E T H Y L E N E BLUE N--Thiazine--Basic-- 1%--25 c.cs. Not toxic, mms. of the stomach not stained with this amount.

9. AZURE I I - -Thiazlne--Basic-- l%--40 c.cs. Mms. of stomach purple. Not toxic.

10. TOLUIDINE B L U E - - Thiazine~Basic-- l%--25 c.cs. Not toxic-stains the mms. of the stomach litt le if any.

11. THIONINE--Thiazine--Strongly--Basic-- l %--40 c.cs. Mms. of the stomach stained bluish. Slightly toxic.

12. T E T R A E T H Y L THIONINE--Thiazine~-Basic-- 1%--40 c.cs. Mms. of the stomach stained slightly purple or bluish. (A sacrifice animal) .

13. T H I O N I N E B L U E - - Thiaz ine- -Bas ic - - l%--25 c.cs. Not noticeably toxic. Mms. of stomach, and the tissues sl ightly stained.

14. BISMARCK BROWN---Azo Group--Basic 1~'~-- 40 c.cs. in a sacrificed animal. Amply through in 2 minutcs. Mms. of the stomach stained brown.

15. CHRYSOIDIN--Azo Gro~ep~Basic--l%--20 c.cs. Through within 5 minutes. Mms. of pouch not noticeably stained.

16. ORANGE G--Azo Group--Acid-- l%--40 c.cs. Mms. colored. Not toxic.

17. CONGO R E D ~ A z o Group--Acid ( I n d i c a t o r ) - - 1%--40 c.cs. Pouch dog. Not toxic. Mms. of stomach

552 AMERICAN JOURNAL OF DIGESTIVE DISEASES AND NUTRITION

stained red. Comes through definitely but not to a great extent.

18. TRYPAN RED--Azo Group- -Ac id- - l%--20 c.cs. Through within 10 minutes, moderate amounts.

19. EOSIN--Xan thene- -Ac id - -1%--40 c.cs. Mms. of the stomach colored pink. Slightly toxic in that amount.

20. MERCUROCHROME - - Xanthene - - Acid - - Not toxic. 1%--20 c.cs. amply through in 2 minutes.

21. E R Y T H R O S I N E - - X a n t h e n e - - A c i d - - l % - - 2 5 c.cs. Mms. stained pink. Not toxic.

22. P H Y L O X I N E - - X a n t h e n e - - A c i d - - 1%--25 c.cs. Mms. of stomach stained deeply pink. Not toxic.

23. ROSE B E N G A L - - X a n t h e n e - - A c i d - - l % - - 2 0 c.cs. Not toxic. Within 5 minutes through, although not to a considerable extent. Mms. of the stomach slightly pink.

24. IOZENE (Commercial germicide) - - Xanthene - - Neutral?--2%--solution of neutral sodium salt of mono- mercurotetra-iodo fluorescein. 10 c.cs. used. About 21~ c.cs. per kilo. Not toxic and came through within 5 minutes. Mms. of the stomach and tissues slightly pink in color.

25. BASIC FUCHSIN--PhenyI Methane---Basic--l% - -10 c.cs. Quite toxic. Stains mms. of the stomach deeply. Continued to be eliminated for 3 days. Seems to have temporarily caused impairment of secretory cells.

26. L ITHIUM CARMINE--Indigo Group---Acid--l% - -40 c.cs. Stains rams. a dark pink. Comes through slightly. Not toxic under ether.

27. E T H Y L CAPRI B L U E N I T R A T E - - O x a z i n - - Bas ic - - l%- -40 c.cs. (Cohen and Preisser) . Mms. of the stomach stained blue. Not toxic.

28. M A L L O P H E N E (W. C. Holmes) (Gall Bladder Dye) (Ma l l i nck rod t ) - - l%- -20 c.cs. Through in less than 5 minutes. Mms. of the stomach not stained. Not toxic.

D Y E S T U F F S E L I M I N A T E D BY T H E S T O M A C H T H R O U G H T H E P A V L O V P O U C H F O L L O W I N G

I N T R A M U S C U L A R I N J E C T I O N

1. N E U T R A L R E D - - A z i n e - - B a s i c - - 1 % - - 25 c.cs. Flank intramuscularly. Through amply within 5 minutes. Mms. of the pouch not stained. Slightly toxic.

2. N E U T R A L V I O L E T - - A z i n e - - B a s i c - - l % - - 2 0 c.cs. Through amply within 5 minutes.

3. M E T H Y L E N E BLUE--Th iaz ine - -Bas i c - - l%- -25 c.cs. Intramuscularly in the flank. Through in small amounts af ter 30 minutes.

4. M E T H Y L E N E BLUE MERCK--U.S .P .X. - -Thia- zine---Basic--1%--20 c.cs. Through definitely in ample amounts within 5 minutes.

5. AZURE I I - -Th iaz ine - -Bas ic - - l%--20 c.cs. Small amounts through within 20 minutes.

6. T O L U I D I N E B L U E - - T h i a z i n e - - B a s i c - - l % - - 3 0 c.cs. Intramuscular ly in flank. Appeared within 15 minutes, definitely, but in small amounts.

7. N E W M E T H Y L E N E B L U E N--Thiaz ine- -Basic- - 2%--25 c.cs. Intramuscularly. Mms. not stained with this amount. Definitely through within 30 minutes. Not toxic.

8. BISMARCK B R O W N - - A z o Group--Bas ic- - l%--30 c.cs. Not toxic. Came through slightly af ter 5 minutes. Mms. of the stomach pouch not stained with such an amount.

9. CHRYSOIDIN- -Azo Group--Bas ic- - l%--20 c.cs. Definitely through within 20 minutes.

10. CONGO RED--Azo Group- -Ac id - - l%--20 c.cs. Definitely through within 5 minutes.

11. EOSIN - - Xanthene - - Acid---1%----40 c.cs. Int ra- muscularly in a sacrificed animal. Through slightly af ter 15 minutes.

12. P H L O X I N E - - Xanthene - - A c i d - - 1% ~ 40 c.cs. Flank intramuscularly into pouch dog. Through in small amounts after 25 minutes.

D Y E S T U F F S N O T E L I M I N A T E D BY T H E S T O M A C H T H R O U G H T H E P A V L O V P O U C H F O L L O W I N G I N T R A M U S C U L A R I N J E C T I O N

All of the dyestuffs tha t had been e l imina ted by the in t ravenous route were t r ied i n t r amuscu la r ly wi th the except ion of Basic Fuchs in , which was too toxic, and E thy l Capri Blue N i t r a t e and Mallophene, which could not be obtained.

1. MAGDALA RED--Az i~e - -Bas i c - - l%- -20 c.cs. Not through.

2. S A F R A N I N O--Az ine - -Bas ic - - l%- -20 c.cs. Not th~'ough.

3. T H I O N I N E BLUE--Thiazine---Basic--2%--25 c.cs. Flank intramuscularly. Mms. of the stomach not stained with such an amount. Not through. Not toxic.

4. SPECIAL M E T H Y L E N E BLUE--Thiaz ine--Basic - - 1 % - - 4 0 c.cs. Not through af ter 20 minutes.

5. THIO NINE--Th iaz ine - -Bas ic - - l %--20 c.cs. Not through.

6. T E T R A E T H Y L THIONINE--Thiaz ine--Ba~ic--25 c.cs. of a 1% solution. Not through af ter 30 minutes.

7. ORANGE G--Azo Group- -Ac id - - l%--40 c.cs. Into flank. Not through af ter 30 minutes.

8. T R Y P A N RED--Azo Gro~p- -Ac id- - l%--20 c.cs. Not through.

9. MERCHROCHROME - - X a n t h e n e - - A c i d - - l % - - 2 0 c.cs. Not through.

10. E R Y T H R O S I N E Xanthene--Acid--1%---40 c.cs. Intramuscularly into flanks. Not through. Not toxic. Mms. not stained.

11. ROSE BENGAL--Xanthene- -Ac ld - - l%-- -20 c.cs. Not through.

12. IOZENE--Xanthene- -Neu tra l? - -2%--5 c.cs. Not through.

13. L IT H IU M C A R M I N E - - I n d i g o Group- - l%--20 c.cs. Not through.

D Y E S T U F F S NOT E L I M I N A T E D BY T H E S T O M A C H T H R O U G H T H E P A V L O V P O U C H

F O L L O W I N G I N T R A V E N O U S I N J E C T I O N

1. TRYPAN BLUE- -Azo Group- -Ac id - - l%--25 c.cs. 2. J A N U S GREEN--Azo Gro~p--Basic--1%,--25 c.cs.

Toxic. Not through. Repeated on an etherized animal with similar results.

3. DIAZINE BLACK (Janus Black)--C.I . 134--Azo Group--Bas ic - - l%--25 c.cs. Quite toxic. Mms. of the stomach pouch not noticeably stained. Repeated twice on sacrificed animals, 1%--40 c.cs. Did not come through.

4. M E T H Y L RED--Azo Group--Weakly A c i d - - l % - 20 c.cs. Not toxic, and mms. of the stomach and tissues not stained.

5. BORDEAUX R E D - - A z o G r o u p - - A c i d - - l % - - 2 0 c.cs. Not toxic.

6. M E T H Y L E N E VIOLET--Thiaz ine--Feebly- -Basic - - 1 % - - 2 5 c.cs.

7. GALLOCYANIN - - Oxazin - - Basic - - 1%--20 c.cs. Not toxic.

8. BRILLIANT CRESYL BLUE--Oxaz ln - -Bas ic - -1% - -25 c.cs. Stained mms. pouch slightly. (?)

9. C R E S Y L E C H T V I O L E T - - Oxazin---Basic--l%--25 c.cs. Not toxic and rams. of stomach pouch not stained with this amount.

10. NIGROSIN--Indul in- -No. 128. W. C. Holmes-- (not a pure dye ) - -Ac id? - - l%- -20 c.cs. Slightly toxic.

11. INDIGO CARMINE-- Ind igo Group of Natural Dyes--Acid---1%--25 c.cs.

12. HEMATOXYLIN- - Ind igo Group of Natural Dyes

Foot No te : 1. I t is a p p a r e n t t h a t m a n y of t he dyes wh ich come t h r o u g h a f t e r

i n t r a v e n o u s in,Jection do not do so a f t e r i n t r a m u s c u l a r in j ec t ion . 2. M a n y of t he dyes a re ind ica to r s and t h e r e f o r e h a v e both ac id a n d

a lka l ine r a n g e s . T h i s is i m p o r t a n t in all of t he se s tudies . 3. T h e f a c t o r of a b so r p t i on of dyes by musc les m u s t be t a k e n i n t o

cons idera t ion .

MORRISON, REEVES AND GARDNER--THE ELIMINATION OF VARIOUS DYES FROM THE PAVLOV POUCH OF DOGS 553

- - ? - - 2 0 c.cs. of 1% solution. Not toxic. Mms. of stomach pouch not stained.

13. VICTORIA BLUE --Diphenylnaphthylmethane--- B a s i c - - l % - - 2 5 c.cs. mms. of pouch not stained. Not toxic.

14. G E N T I A N V I O L E T - - T r i p h e n y l Methane--Basic - - 1 % - - 2 5 c.cs. Tissues not stained and not toxic.

15. E T H Y L V I O L E T - - T r i p h e n y l M e t h a n e ~ B a s i c - - l % - -25 c.cs. mms. not colored. Not toxic.

16. M E T H Y L B L U E - - T r i p h e n y l Methane--Acld 1% --25 c.cs.

17. LIGHT GREEN--Tr~phe~yI Methane- -Ac id - -2% - -25 c.cs. Not toxic and does not come through. Stains tongue but not much of stomach.

18. A N I L I N E BLUE- -Tr ipheny l M e t h a n e ~ A c i d - - l % - -25 c.cs. rams. of pouch not stained.

19. PYRROL BLUE--T~ipheny l Methane--Acid 1% --25 c.cs.

20. B R I L L I A N T G R E E N - - T r i p h e n y l Methane--Basic - - 1 % - - 2 5 c.cs. Not toxic, and mms. of the pouch not stained.

21. A U R A M I N E - - ( W . C. Holmes) C.I. No. 655-- Diphenyl M e t h a n e - - B a s i c - - l % - - 2 0 c.cs. Not toxic, and mms. of the stomach pouch and tissues not stained.

22. A U R A M I N E G - - ( W . C. Holmes) C.I. No. 656-- Diphenyl M e t h a n e - - B a s i c - - l % - - 2 0 c.cs. Not toxic. Doesn't stain mms. of pouch or the tissues.

23. P Y R O N I N - - X a n t h e n e - - B a s i c - - l % - - 1 0 c.cs. Toxic, not through. Repeated using 40 c.cs. of 1% solution. Mms. of stomach and the tissues stained slightly on a sacrificed animal. Doesn't come through.

24. E R Y T H R O S I N E (AI. Sa l t ) - -Xan thene - -A c id? - - I %--20 c.cs. Not toxic. Mms. not stained. Repeated, 1% - -40 c.cs. Not through. Doesn't go into solution readily.

25. P H E N O L S U L P H O N E P H T H A L E I N - - X a n t h e n e - - Ac id - - ( Ind i ca to r ) - -1%- -25 c.cs.

26. THYMOL BLUE - - Xanthene- -Acid- - ( Ind ica tor ) - - 1 % - - 3 0 c.cs. into a pouch dog. Mms. not colored. Dye is toxic.

27. BROM CRESOL PURPLE--(DibromcresolsuIpho- nephthalein) ~ Xanthene - - A c i d - - (Indicator) - - 1 % - - 2 0 c.cs. Not toxic and mms. of the stomach pouch not stained.

28. BROM THYMOL BLUE--(Dibromthymolsulpho- nephthalein) - - Xanthene - - Acid - - (Indicator) - - 1 % - - 2 0 c.cs. Not toxic, and mms. of the stomach pouch not stained.

29. CRESOL R E D - - (O-Cresolsulphonephthalein) - - Xan thene - -Ac id - - ( Ind i ca to r ) - -1%- -20 c.cs. Did not stain rams. of the stomach pouch. Not toxic.

30. CHLOR P H E N O L - R E D - - (Dichlorphenol)--Xan- thene- -Ac id- - ( Ind ica tor ) - -Did not stain mms. of the stomach pouch or the tissues. Not toxic.

31. F L U O R E S C E I N - - Xanthene - -Ac id~- l%- -40 c.cs. on a sacrificed animal. Mms. of the stomach not stained with this dosage. It did not come through.

32. ISAMINE BLUE--Oxaz ine - - -Bas ic - - l%- -25 c.cs. 33. BERLIN B L U E - - L a - - U n c l a s s i f i e d - - l % - - 2 5 c.cs.

Tissues not stained. 34. PONTAMINE SKY B L U E - - ( 5 1 3 ) - - 5 BX - -U n-

classified--! ~ - - 2 5 c.cs. 35. P O N T A M I N E SKY BLUE--AX--Unc la s s i f i ed - -1

%--25 c.cs. 36. P H E N O L I N D O P H E N O L - - Unc lass i f i ed - - l%--25

c.cs. Mms. of pouch not colored. 37. T O L U L E N E B L U E - - T h i a z i n e - - B a s i c - - l % - - 2 5

C.CS.

38. P O N T A M I N E B L A C K - - E X - - U n c I a s s i f i e d - - 2 ~ - - 25 c.cs.

39. BENZAMINE R H E I N B L A U - - ( 5 1 3 ) - - A z o - - Basic-- l%---25 c.cs. Not toxic. Mms. of stomach pouch not noticeably stained.

40. NATIONAL ALKALI BLUE- -2GP . CI-710-- Unc la s s i f i ed - - l~ - -25 c.cs. Not toxic and rams. of the stomach not stained.

41. NATIONAL ALKALI BLUE--4GP--Unclass i f ied - - 1 % - - 2 5 c.cs. Not toxic and mms. of stomach pouch not stained.

42. P O N T A M I N E G R E E N - - Unclass i f ied-- 1% - - 25 c.cs. Not toxic. Mms. of the stomach pouch not stained.

43. J A N U S B L U E - - U n c l a s s i f i e d - - l % - - 2 5 c.cs. Mms. of stomach pouch not colored. Slightly toxic. Repeated with confirmation on sacrificed animals, 1%--40 c.cs.

44. L E U C O F A S T G R E E N - - F C F - - U n c l a s s i f i e d - - l % - -25 c.cs. Not toxic, did not stain mms. of stomach pouch nor the tissues.

45. INDIGO M O N O S U L P H A T E - - U n c l a s s i f i e d - - l % - - 30 c.cs. Not toxic. Mms. of stomach pouch not stained.

46. INDIGO T E T R A S U L F O N A T E - - Unclassified--1 %--30 c.cs. Mms. of stomach pouch not stained. Not toxic.

47. INDIGO D ISU L FO N A T E - -U nc l a s s i f i ed - - 1%- - 30 c.cs. Mms. of stomach pouch not stained. Tissues slightly stained.

48. DIANIL Y E L L O W - - R - - C . I . = 6 4 9 - - W . C . H . - - U n - classified--l(/~--25 c.cs. Rather toxic. Did not stain rams. of stomach pouch.

49. RHODULIN VIOLET- -Unc la s s i f i ed - - l%- -15 c.cs. Quite toxic. Mms. of pouch and mouth pink--skin pink.

50. ALIZARIN SAPHVIOLET--1187- -Unclass i f i ed- - 1C~--20 c.cs. Toxic. Mms. of the stomach pouch not stained. Not toxic.

51. PONCEAU de X Y L ID IN E - -U nc l a s s i f i ed - - l%- - 20 c.cs. Not toxic. Mms. of the stomach pouch not stained.

52. T A R T R A Z I N E - - (NAC)- -W.C.H. - -Unc lass i f i ed- - 1%--20 c.cs. Not toxic and not through. Mms. of stomach pouch not stained.

53. PONTACYL LIGHT YELLOW--GG--Unclass i f ied --lC/~--20 c.cs. Mms. of stomach pouch not stained. Not toxic.

54. DIANIL YELLOW--3G- -C . I . 647--Unclassif ied-- 1 % ~ 2 0 c.cs. Mms. of the stomach pouch not stained. Tissues not stained. Not toxic.

D Y E S T U F F S NOT E L I M I N A T E D BY T H E S T O M A C H T H R O U G H T H E P A V L O V P O U C H

F O L L O W I N G A D M I N I S T R A T I O N V I A S T O M A C H T U B E

1. Aqueous solutions, 1 g r a m per hundred of dis- t i l led water , in amounts of 20 c.cs. were admin i s t e red by s tomach tube to pouch dogs. All of the dyes were used which had been shown to be e l iminated a f t e r in t ravenous admin i s t r a t ion , wi th the exception of th ree dyes : Iozene, Basic Fuchs in and E thy l Capri Blue Ni t r a t e . Iozene and Ethy l Capri N i t r a t e could not be obtained in f u r t h e r supply and the basic fuchs in was fe l t to be too toxic fo r considerat ion. None of the dyes were e l imina ted in the pouch a f t e r an hour following the admin i s t ra t ion .

2. Neu t r a l red and Neu t ra l Violet were then separ- ately t r i ed by s tomach tube in alcoholic solutions. One g r a m of each per 100 c.cs. of 70% alcohol were used, 20 c.cs. being the amount admin i s t e red in each case. These dyes only were used, because it was fe l t t h a t they were e l imina ted as quickly by the s tomach as any of those t r i ed in t ravenous ly and in t ramuscu la r ly . An hour fo l lowing admin i s t ra t ion , ne i the r had appeared Jn the pouch.

F o o t N o t e : 1. " 'T issues s t a ined o r not s t a i n e d " r e f e r s to comple te au tops i e s which

offered the o p p o r t u n i t y of e x a m i n i n g t i s sues ( o r g a n s ) in genera l . 2. I f the con ten t s of the Pav lov pouches had been d ropped in to bas ic

solut ions v a r i o u s dye colors m a y h a v e a p p e a r e d i n d i c a t i n g secre- t ion in a leuco f o r m . I n o t h e r cases t r e a t m e n t w i t h a weak ly ox id iz ing solut ion m a y l ikewise h a v e produced color i n d i c a t i n g reduc t ion of dye in musc le or o the r t i ssues .


DYESTUFFS COMING THROUGH GASTRIC MUCOSA (POUCH) AFTER RECTAL

INSTILLATIONS 1. These results have not been satisfactory or con-

stant up to the present time. 100 c.cs. of 1% neutral red instilled into rectum had come through the pouch after 12 hours. In some instances it appeared faintly 40 minutes after injection. This work is to be repeated.

DYESTUFFS COMING THROUGH GASTRIC MUCOSA (POUCH) A F T E R I N T R A P E R I -

TONEAL INJECTIONS 1. Those dyes which came through after intraven-

ous and intramuscular injections were tried. These results have not been satisfactory up to the present time. The rectal and intraperitoneal routes are mentioned now because the former has not been tried here- tofore while the latter has had only limited trial.

Basic fuchsin appeared to be a toxic dye and after its use there was no secretion of HCI. The animals in which this occurred and two other animals which had developed marked hypochlorhydrias approaching achylias were utilized fvr the injection of dyes which were known to be eliminated through the gastric mucosa. These experiments are to be repeated. How- ever, it is interesting that methylene blue 1%, 25 c.cs., was excreted only in the smallest amount (there was a small amount of HC1). Toluidine blue, 1%, 25 c.cs., was injected into the oldest collie dog in which little HC1 was excreted and no dye appeared.

REMARKS 1. 1% solutions (1 gram per 100 c.cs. of water)

were employed. The dyes did not always dissolve 100% in the solvent; a moderate amount of heating was necessary in some cases. In those instances in which a solution of 1 gram to 100 c.cs. could not be approxi- mated or in which solution was brought about with difficulty the dye was omitted or if used attention is called to the solubility difficulties.

2. In the future the authors recommend that in- stead of saying the mucousmembrane of the stomach pouch was or was not stained, a thing difficult to be certain of, it would be better to compare the heavy staining of the mucous membranes by such dyes as lithium carmine and phloxine with that of methylene blue, which stained them very little; such could be designated as (4 plus), vs. (p lus-- ) . To establish a standard intensity of staining requires fur ther work and for that reason we are not prepared to offer this simplified scheme at present.

3. "Unclassified dyes" refers to those dyes for which reference works (Schultz, Conn) offer unsatis- factory information regarding their groups and acidity or basicity.

4. Disagreement with Kobayash i : - - (ou r work is in disagreement with many previous investigators but since Kobayashi has made the more recent and extensive studies we call attention to these differences so that the importance of the present study is under- stood).

1. He reports Janus Black being eliminated by intravenous injection. This did not occur with us, even though it was 3 times repeated, using as high as 40 c.cs. of a 1 gram per 100 C.CS.

2. Several dyes, acid, which he reports as not being eliminated following intravenous injection were found by us to be eliminated quite definitely, namely the following: 1. Congo Red 2 . Eosin 3. Erythrosine 4. Lithium Carmine 5. Phloxine 6. Trypan Red (and among the basic dyes) 7. Fuchsin

5. Basic dyes do seem to be the ones most suited for elimination, particularly is this so in the intramuscular injections, these being predominantly the basic dyes, and the dyes most quickly and abundantly eliminated. Neutral Red, Neutral Violet and Methy- lene Blue are basic dyes. However, the degree of basicity is not proportional to elimination as is shown by Thionine (a strongly basic dye), neutral red and neutral violet (weakly basic) and the other basic dyes.

For the purpose of clarity the following table of Conn showing the grouping of dyes is appended. Dyes of the Nitro, Azo and Oxyquinone Groups:

1. The Nitro Group Picric Acid Martius Yellow Aurantia

2. The Azo Group Orange .G Bordeaux Red Janus Green B Fast Yellow Methyl Orange Orange IV Orange I Narcein Amaranth Sudan III Sudan IV Biebrich Scarlet, Water Soluble Bismarck Brown Y Congo Red Trypan Red Benzopurpin 4B Trypan Blue Chrysoidin R Chrysoidin Y Others

3. Oxyquinone Group Alizarin Alizarin Red S Purpurin

The Quinone-Imide Dyes 1. The Indamins

Toluylene Blue 2. The Thiazins

Thionin Methylene Blue Methylene Azur Methylene Violet Methylene Green Toluidine Blue 0

3. The Oxazins Brilliant Cresyl Blue Nile Blue Sulphate Cresyl Violet

4. The Azins a. Amido-Azins or Eurhodins

Neutral Red Neutral Violet

I~¢IoRRISON, REEVES AND GARDNER--THE ELIMINATION OF VARIOUS DYES FROM THE PAVLOV POUCH OF DOGS 555

b. Safranins Safranin 0 Amethyst Violet Magdala Red

c. The Indulins Nigrosin, Water Soluble

The Phenyl Methane Dyes 1. Di-Phenyl Methane Derivatives 2. Tri-Phenyl Methane Derivatives

a. Di-Amino Tri-Phenyl Methanes Malachite Green Brilliant Green Light Green SF, Yellowish

b. Tri-Amino Tri-Phenyl Methanes (Rosanilins) Parafuchsin Rosanilin Basic Fuchsin New Fuchsin Acid Fuchsin Hoffman Violet Methyl Violet Crystal Violet Gentian Violet Methyl Green Iodine Green Spirit Blue Methyl Blue Amilin Blue Others

The Xanthene Dyes 1. The Pyronins

Pyronin G Pyronin B

2. The Rhodamines Rhodamine B

3. Fluorane Derivatives Fluorescein (Uranin) Eosin, Y (i.e. Yellowish) Methyl eosin Ethyl eosin Eosin, Bluish Erythrosin, Bluish Phloxine Rose Bengal

4. Phenolphthalein and the Sulphonphthaleins Phenolsulphonphthalein (phenol red) Brom Cresol Purple Thymol Blue Others

The Natural Dyes The Indigo Group Indigo (Blue) Indigo Carmin Litmus ttaematoxylin Others

Classification of important biological dyes based upon chromophores (Conn). (They can be fur ther grouped into acid and basic chromophores).

1. The nitro dyes e.g. picric acid

2. The Azo Group e.g. methyl orange, Bismark brown, orange G, Congo red, Sudan III and Sudan IV

3. The Oxyquinone Group e.g. alizarin

4. The quinine-imide group, including a. Indamins b. Thiazins; e.g., thionin, toluidine blue, methy-

lene blue c. Oxazins; e.g., brilliant cresyl blue, Nile blue

d. Azins, including (i) Amido-azins; e.g., neutral red (ii) Safranins; e.g., safranin O, magdala

red (iii) Indulins; e.g., nigrosin

5. The phenyl-methane dyes, including a. Diphenyl-methanes, e.g., auramin b. Diamino tri-phenyl methanes; e.g., malachite

green, brilliant green, light green c. Triamino tri-phenyl methanes; e.g., basic

fuchsin, acid fuchsin, methyl violet, gentian violet, methyl green, anilin blue

d. Hydroxy tri-phenyl methanes (Rosolic acids) ; e.g., aurin, corallin red

6. The Xanthene dyes, including a. Pyronins; e.g., pyronin G and B b. Rhodamines; e.g., Rhodamine B c. Fluorane derivatives; e.g., eosins, erythrosin,

rose bengal d. Phenolphthalein and the Sulphonephthaleins

DISCUSSION

No attempt will be made at present to review the literature on the subject of dyes. I t is voluminous and the reviews are many. Only a few of the more recent contributions will be discussed since they are necessary for the sequential development of the problem at hand.

After the work of Fuld and Finkelstein, Hirabayashi thought it of interest to observe the excretion of neutral red under various produced pathological conditions. In normal stomachs he observed, through gastric fistulas, the appearance of neutral red after the administration of an Ewald meal, calcium carbonate, histamine, pilocarpine and alcohol. He concluded that the speed with which neutral red appeared depended upon the intensity of secretion of the secretory glands. He now washed the stomach with ~ % silver nitrate solution and found that af ter observations extending over 2 hours no dyestuffs appeared in the stomach though the HC1 secretion was persistent. After 48 hours the stomach again secreted normally following histamin and dye injection. From these and similar experiments it appeared to Hirabayashi that a local damage to the gastric mucosa diminishes the intensi~y of secretion and leads to a reduction in dye excretion over that shown for the normal stomach. The stimula- tion with silver serves also to show that the glands were not able to excrete dye but that the secretion of HC1 was not interrupted. It is therefore possible, Hirabayashi concludes, that the dye is excreted through the pepsin glands. We repeated this experi- ment and are in disagreement as shown by the following data.

E L I M I N A T I O N OF DYESTUFF FOLLOWING INTRAVENOUS INJECTION A F T E R THE

STOMACH HAD BEEN TREATED WITH SILVER NITRATE SOLUTION

Ninety c.cs. of ~ % solution of silver nitrate was ad- ministered to a pouch dog by stomach tube, and the pouch was washed with ~ % silver nitrate solution. Ten minutes later 20 c.cs. of a 1% solution, aqueous, of neutral red was injected intravenously. Within 2 minutes the dye appeared from the pouch and in the stomach as amply as had been found previously. The same results were obtained with the stomachs of white rats.

Our results are entirely different from those of Hirabayashi, and they corroborate other conclusions


which we have been able to reach regarding the mechanism of neutral red excretion. This mechanism is of fundamental importance in the study of certain diseases as we hope to point out in future communica- tions.

Dawson and Ivy studied the elimination of dyes by the gastr ic mucosa. They used dogs with an isolated fundic pouch (Pavlov Pouch) and dogs with an isolated pyloric pouch, the lat ter animals being used to ascertain if certain dyes were eliminated by the mucus cells. Of th i r ty- three dyes studied, thir teen were eliminated in the gastr ic juice. However, we were unable to corroborate these results in their en- t irety. Dawson and Ivy report a relationship between the amount of dye eliminated and the ra te of secretion, i.e., the more gastr ic juice, the more dye eliminated. On the other hand, they report the elimination of neutral red through the pyloric pouch when the mucosa of the pouch was activated with gastr ic juice. I t seems hardly possible tha t this was actual elimination, because we have been able to demonstrate a selective secretory mechanism for neutral red. Dawson and Ivy could not predict the effect of a dye f rom a study of its chemical constitution but they mention selective secretion as an obvious factor that must be considered in dye elimination, that is, some dyes may possibly be eliminated through the parietal cells, others, through the chief cells or mucus cells. Several dyes, of which neutral red is the best example they write, can be de- tected by microscopic examination to be passing out through the parietal cells as the secretion in the canali- culi is rendered conspicous by the dye. I t would be difficult to correlate this finding with the elimination of neutral red through the pyloric pouch since the pylorus (unless the pouch included some oxyntic cells) contains no parietal cells. Dawson and Ivy believe that under certain conditions neutral red though most readily eliminated by the parietal cells, can be eliminated by the mucous cells of the stomach. They conclude finally tha t the parietal cells are the cells chiefly con- cerned in the elimination of dyes by the gastr ic mucosa and whether or not a dye is eliminated by these cells depends on some unknown as yet physical ra ther than chemical proper ty of the dye as there is no chemical proper ty characterist ic of the dyes that are eliminated. Fur the rmore it seemed to them tha t the problem of dye elimination by the gastr ic glands has a grea ter scientific import than practical value. By comparing our Table with their ' s the importance of repeating these dye elimination tests becomes apparent. For example in our tests neutral violet came through amply, in the tests of Dawson and Ivy the result was negative. Moreover, the extensive reports of Kobaya- shi are not entirely corroborated by our investigations and attention has been directed to some of the differences.

Kobayashi has studied the absorption, secretion and excretion of dyes perhaps more extensively than any other recent investigator. He cannot agree with the conclusion of other invest igators tha t the mechanism of the elimination of dyestuffs into the stomach could be explained simply by diffusion or tha t it depended on the gastr ic secretory function. He also disagrees with Hirabayashi ' s conclusions for he believes it must be the gastr ic glands and especially the parietal cells that secrete the dyestuffs. Matsuo as well as Glaessner and Wittgenstein, the lat ter in histological studies of neutral red elimination, support Kobayashi in this fat-

ter opinion. Kobayashi 's experiments were very well controlled and minutely observed and he was able to correlate the concentration of eliminated dyes, hydrochloric acid and the pepsin. His experiments also con- firmed the lack of relationship between the diffusibility of dyestuffs and their intensity of elimination; the lipoid solubility of dyestuffs did not explain their elimination into the stomach. Acid dyestuffs in general, were found not to be eliminated into the stomach even when soluble in lipoids. Our work would not support this observation although we too, found that basic dyes were predominant among those passing through the gastr ic mucosa. Kobayashi investi- gated 65 acid dyes; all of them had different proper- ties physico-chemically or biologically, that is, in chemical construction, in diffusibility, in solubility in lipoids, or in vital staining, but none of them were perceived in the gastr ic juice.

Kobayashi concludes that the dyestuffs which are eliminated into the stomach are chlorides of dye bases and have amidogroups or their alkylsubsti tutes in their auxochrome. They belong to the azo, azine, oxazine, thiazine or acridine group and the i r chromogens have all - - N = in their chromophores and great ly resemble the fundamental unit of protein. They have some diffusibility and all have a positive charge. Kobayashi points out the resemblance in construction between these chromogens and the fundamental unit of protein and consequently he continues, "the dye bases of these dyes may be considered to have a construction similar to protein itself in some points." He suggests that this may be the reason why only the dyes of these groups can be eliminated into the stomach. Finally the elimination of dyestuffs is explained by a mechanism similar to the secretion of hydrochloric acid; he shows that his explanation harmonizes very well with the fact that when the parietal cells secrets HC1 in a high concentration, they secrete dyestuffs in a relatively large quantity. The concentration of pepsin did not run parallel to that of the dyestuffs as exactly as the acidity. When the concentration of hydrochloric acid in the gastr ic juice is increased by a pilocarpine injection, the concentration of dyestuffs eliminated into the stomach is also increased and when the fo rmer is decreased by atropin, the lat ter is also decreased. Also with patients, Kobay- ashi found the curve of concentration of neutral red eliminated into the stomach a f t e r its adminis t ra t ion into the blood circulation generally to run parallel with that of hydrochloric acid in the gastr ic juice. The work is indeed convincing and we have been able to corroborate it except that in our dye tests several acid dyes were found to be eliminated through the gastr ic mucosa. This finding necessitates another explanation of the mechanism of dye elimination than tha t offered by Kobayashi. The important fact however is the verification of the physiological mechanism of dye elimination. Knowing this, it becomes applicable for clinical use especially as a diagnostic measure to say nothing of the interpretat ion of pathological change which it allows. Fur the r development of this topic will come later.

In 1932, Henning reported his work on the excretion of dyestuffs through the gastr ic mucosa of frogs. This work was based on some earlier clinical work which Henning, Jurgens and Orstein did in collaboration in 1931. The experimental a r rangement was unique in that frogs, the gastric mucosae of which are almost

MOI~RISON, REEVES AND GARDNER--THE ELIMINATION OF VARIOUS DYES FROM THE PAVLOV POUCH OF DOGS 557

completely transparent were used while under the quieting influence of urethan. The stomach of the narcotized frog (urethan) was opened at the greater curvature and by an appropriate technique the excretion of a given dyestuff from the stomach could be followed visually in all its phases. The results obtained are remarkable when compared with Dawson and Ivy's and Kobayashi's for af ter testing a long series of dyestuffs it was shown that only acid dyestuffs are excreted into the stomach, provided they are in suffi- ciently fine dispersion; basic dyestuffs are not so excreted. As an example, the microscopical study of neutral-red excretion shows that the indicator in red color first appears on the surface of the mucosa, while it passes through the mucosa as neutral yellow. Ac- cordingly, the gastric hydrochloric acid is first formed on the covering epithelium. The dyestuff excretion Henning reports "is a function of the fundus glands; the antrum glands do not participate." Dawson and Ivy reported neutral-red excretion through a pyloric pouch in the dog. The excretory function of the gastric muccsa, Henning concludes, is connected with the secretory function. It can be elicited in secretory rest by means of secretory stimulants (histamin). In other words, the excretory function of the gastric mucosa is closely related to the secretory function; it stops on secretory rest and can be elicited through histamin. Uranin (fluorescein), eosin, erythrosin (all acid xanthene dyes) neutral red (weakly basic azin), Congo red (acid azo) magdala red (basic azin), t rypan blue (acid azo) and methylene blue (basic thiazin) were a few of the dyes which came through the gastric mucosa. It will at once be seen that 3 of these dyes are basic which makes it difficult to understand the conclusion that only acid dyes are eliminated, except that Henning refers to neutral red as an indicator dye and therefore acid as well as basic. Furthermore, except for Henning there is general agreement that t rypan blue is not eliminated through the gastric mucosa. We found that it was taken up by the phagocy- tic cells but was not in the glanduler cells. Moreover, in our work t rypan blue was injected intravenously into a rabbit and followed by neutral red; the latter appeared through the gastric mucosa but the trypan blue did not. Henning finds that trypaflavin is taken up by entirely different cells than uranin. The dyes mentioned above all electively begin to stain the capil-

lary walls, the submucous connective tissue and the glandular cells. Henning recalls the extensive experimentation on albino rabbits of F. P. Fisher who showed that the different behavior of the dyestuffs is determined by their colloid constitution and that the fate of dyestuffs which have been introduced into the blood current, is governed by the degree of dispersion, the electric charge and surface tension phenomena.

SUMMARY

In order to furnish a working basis for fur ther investigations it has become essential to repeat the study of dye elimination through the gastric mucosa. With- out reviewing the voluminous literature, three repre- sentative and rather recent papers are examined to show how extremely varied the results have been. Often the differences are so extreme that reasons are sought to explain them either in the type of experimental animal, the method of experimentation or the interpretation of results. I t becomes apparent that our interpretations explain some of the incongruities but there still remains a fundamental difference in results. All are agreed that it is the fundus mucosa which eliminates those dyes which are excreted. We too, have reached this conclusion but furthermore, we have found that both acid and basic dyes are eliminated (more of the latter) which differs from the results obtained by the other investigators. Many dyes may be both acid and basic. In fact, the acid or basic reac- tion of many dyes depends on the nature of the sub- groups which confer water solubility upon the mole- cule. Our work corroborates parts of each of the other investigators but not enough to substantiate any of their explanations of the mechanism of dye elimination nor the reasons for selective elimination. It would seem that size of dye particles apparently plays little role since it has been shown by the ultrafiltration experiments of Allisbaugh and Hyde that methylene blue, a dye readily eliminated, is considerally larger in par- ticulate size than a dye definitely withheld. Careful study of the present investigation leads us to the conclusion that it represents a reliable basis for the studies which we are pursuing but it does not allow us, as yet, to explain either mechanically, physiolo- gically or chemically dye elimination by the mucosa. This we hope to do in a future communication.

R E F E R E N C E S 1. Al l i sbaugh, H. C., and Hyde, R. R . : F r a c t i o n a l U l t r a f i l t r a t i on .

Am. Jour. Hygiene, 2l :64, 1935. 2. Conn, H. S . : Biological Sta ins . Publ ished by the Commiss ion,

Geneva, N. Y., 1929. 3. Dawsoi~, A. B., and Ivy, A. C. : Cont r ibu t ions to the Physiology of

Gas t r ic Secretion. VI I . The E l i m i n a t i o n of Dyes by the Gas t r ic Mucosa. Am. J. Phys., 73:304, 1925.

4. F inke ls te in , R. : Zur F r a g e der Fa rbs t a f f aussche rdung durch den Magen. Arch. f. Verdauungskr., 30 :~99. 1923.

5. Fuld, E. : Aussche idung von Neu t rn l ro t a u f den Magen. M~dnchen. reed. Wochenschr., 43:908, 1908.

6. t t e n u i n g , N . : Die Aussche idung von Farbs tof fcn durch die Magen- schle imhaut . Microscopic I nves t i ga t i ons on L i v i n g Animals . Arch. fur Espy. Path. and Pharmakol., 165:197, 1932.

7. Hi rabayash i , N . : Exper imenteI Ie Un te r suchungen Zur Chromodi- agnos t ik der Sekre t ionss t~ rungen des Magens. Archiv. fur Verdauun~skr., 33:71, 1924.

8. Kobayashi , K. : Expe r ime-¢a l Studies on Absorpt ion, Secret ion ~nd Excre t ion . On the Secretion ~nd the Absorp t ion of Dyestuffs by the Stomach. I. On the Secretion of Dyestuffs in to tbe Stomach. A('ta Scholae Mcdici*lalis. 8:465, 1926.

9. SchuItz, G. : Farbstoff tabe!len. Leipzig , 1931.

the elimination of various dyes from the pavlov pouch of dogs

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