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OJ- lOKONTC^

i'll\'sii)i.in;u \i, SI Kiis

S' ,5- ^^N rill-: l-OK.MATION Oh' HYDROCHI.ORIf ACID
IN I UK I.ASTKIC Tcia'I.KS Ol- THE \ ERTKBRATli
s:i'MAl II. i'.\ I H loiirr

THh I NIVERSITY LIBRARY: PUBLISHED BY
THL LIBRARIAN. 1020

'Qlntfereltt of Toronto StuMet

COMMITTEE OP MANAGEMENT

Chmrmtm: Sii Rosnrr Auxanoui Faixohu, LL.D., K.C.M.U

PraiidMit of tli* UMYcnity

PsorasMi W. J. ALSXANDn, Pm.D.

Propsmor J. J. Maciunzib, B.A., M.B.

Propbmor J. P. McMuRRicH, Ph.D.

Bric-Gkn. C. H. Mitchrix, B.A.Sc., C.B.,C.M.G., D.S.O.

Propruor G. H. Nrrolrr, Ph.D.

PROPRtaoR Groroe M. Wromo, M.A.

QmmmlSdUtr: H. H. Lanotom, M.A.

Librarian of tb« Uaivartity

ON THE FORMATION OF HYDROCHLORIC ACID IN
THE GASTRIC TUBULES OF THE VERTEBRATE
STOMACH

BY

J. B. (oil IP, M.A.. I'li.l).

fUmmmmmmmm

ON THE

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Since
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al aspect
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iiNTHK FORMATION OF nYf)ROCHIX)KIC ACID IN THK
GASTRIC TUBULES OF THK VERTEBRATE
STOMACH

I. LiTKRATURE

Since the time it wa» first rccofrnized that hydrochloric acid
vM normally produced during digeation by the activity of the
ifastric niuctma, numerous attemptH have been made to solve
the physiological problem therein involved. The purely chemi-
al aspect of the problem, the formation of a free mineral acid
fritm an alkaline blood plasma, U-d many observers at differ-
• nl tmies to seek to discover the mode of formation and seal
"f origin of this acid, using for this object methods of inves-
ngation which we might term purely chemical. Others who
«irc engaged in working on the histological structure of vari-
<M* parts, found, that, in the case of the stomach, their de-
'MU'ii morphological de.scriptions of the gastric mucosa afTord-
-^d them ample material as a basis for lengthy speculations as
tothe correlations of the structures studied, and of the physio-
"Sfieal processes known to be associated in one way or another
with these structures. The literature, therefore, can be
placi'd under two heads, the first dealing with the histological
dt'taiis and the speculations as to the function resulting there-
from; the second with the direct attempts to determine the
place of origin of the acid itself. A summary of this litera-
ture under these heads, and in the order given, follows.

1. The Mari)hological Data and the Disctuisiona Thereon

The Mammalian Stomach. The Mammalian stomach dif
fers from that of all other Vertebrates in that in the gastric
tubules of the fundus region two quite separate and distinct
types of gland cells are manifested. These have now long been

.1

I

I

3 i
2 i

COLLir: F0«MATU>S or HVfMliKHUHlIC At It)

known ri»ip«'ctlvi'ly ai the chief or pt'ptic r«tlU and th«« n«ni t^i
or Ixirdi'r cfll«. Thi* duality dinplttytni In th«' •truitur»' < f th.
Mammalian pvpUr glanda or labdru«<n wa« fint drat nUd hs
liftdi-nhain (IM7h). ||t> iiamt'd Iho two form* which h.' (ml
obnTved th»' "llauptx«>ilen" and thf "IU'l«'gi«>lli'n" rf«|M'.ti\iH

8incit that timi' much diacuaaion as to the natiir** and %in
nincanco «)f »>ttch of thtw two formn of irland o-IIb haw t,jk. n
placf. Th»> writing* in thia connwtion havi- bwn v«'ry num. t
ouit, and thi' authom havt- um-d varioiu qtiahfyinit tirnH in
thfir dfscriptiona of \.hv»v cell*. The chiff c«>lla or Haui.t
ifllen have bwn apokcn of aIro aa "adetomorphe Ztlldi,
"polygonale Zellen," "central t-eJli," "cellea principiiU.
"Lkhlfimzelk'n." et'-. The parietal eells. or BeiegzelUn, hav.
also been de?"ribed aa "delomorphe Zellen." "Pepainzollen '
"Kegelformigezellen," "border cells" and "Cellule riru-
prenti."

The great dilTerence in size, in optical behaviour and in thi
ataining rapacities of the two types observed by Heidcnhuin
led him to the conclusion that the chief cells had to do with
the elaboration of the proteolytic ferment pepsin, and that thi
parietal cells supplied the greater part of the fluid of thi' fi. ■
trie juice, and were also associated with the formation of ti
hydrochloric acid. Heidenhain also noted that the pari<t.i
cells were relatively more abundant in the upper third of th.
bodies of the gastric tubules of the fundus region. This, hr
thought, was very signiflcant, as he had observed that i\-
tracts made from the deeper layers of me gastric mm nsa had
greater peptonizing power than similar extracts which h.id
been prepared from the more superficial layers. Eb.stoin atui
Crutzner (1872 and 1871) obtained sim.'ar results. They con
eluded that the greater part of the pcpsii was probably elabir
ated by the chief cells. They advanced the view that the paru -
tal cells were rich in the chlorides of the alkalies and by .sor,'
unknown process liberated the free acid from these salts.

Nussbaum (1878) stated that the parietal cells stain-i
more deeply with osmic acid om^ hour after the onset of ai '
ity than at any other time. He held to the view that the pam -

('ol4.ir: FUKMATION r»r IIVORfM lll.iMli- A«'tf>

^

4I (t'll» ['"""Hurfd tho iM'pmn. thf chuf (i.|U nlonir with tht-
inlorlc gland a-lln Ih-iiik rTiufJn tiriMluffm, lii> *vj»ii aluu of
.If opinion Ihut ih»' pAripUl i-flU wori- homologou!* with thi-
'■ptir Kl«nd cclU of th«' lown Vi-rU-braU. which, ihi-rifori-.
«' iild prixlun' [H'piiin.

Kdinget" ( IM7I0 htltl that h wiMratf function for fm h t>p««
I ..'II could not \h' di'rtnitcly proven.

UrgUy rtnd ScwhII ( |M7;»» Hnd Utngliy ( IHMI) sluditd the
iarrf«>s during funrtional rent and activity in th«' gasitric
il.uU'ji. and found that in tht- chu-f (.■llii, when th»»
iUiuljt wiTc m-t HccKling, wi'ii" griinulf* which began to
ii»;ip|Hiir or to become fewer in numlM-r when secretion be-
inn. A« the pepsin <if the ^.Tretion or extractible front the
r.iicona «'ither during r.-st or at any stage of activity wa.n
ippaiently in proportion to the number of gran;ile.>i pres. .it it
va.H claime<l that the granulen themselves were compo.HmJ of
th. mother substanc (://»;/•.;/.») of the ferment pepsin and
that, therefore, the chief cells .secreted pepsin. The parietal
"Ws. on the other hand, were found free from granules during
;hi' resting stage and developed very f^ne ones during activity
*hi.h. however, did not appear to be associated with the lur-
niiitiun of any ferment. These cells were observed to become
iarifcr in size as the secretion began, and they remained en-
arjred as long as secretion continued. This made it ••vident
that they .secrete some constituent of the gastric juice. A.s the
parifta! cells are limited in their distribution to the cardiac
ptirtion of the stomach aiid as this portion alone furni.'^hos an
and .<iecretion the authors concluded that the parietal cells are
nnci'med in the secretion of the hydrochloric acid of the gas-
tric juice. I.Angley (1H«1) in constHjuence applied the term
"iinitic (i.e., acid-forming) to these celLs.

(Ireenwood (1885) found that the parietal cells .stained
iuitc markedly with nitrate of silver solution. This was prob-
ably the first direct evidence of their function.

Oppol (1806) maintained that a perfect functional division
wa.s impossible. There could only h> a quantitative difTerence
>n the relative amounts of the constituents of the gastric juice

J

Cou.iF : Formation of Hydrochloric Acid

contributed by each type of cell. The parietal cells, he thought,
were represented by the body cells of the fuadus gland tubules
of the lower Vertebrata, while the homo' les of the chief
eel'" wero to be found in the neck cells of i.ie gland tubules of
these lower forms. Neither type of cell was a new departure
in the Mammal, he claimed.

Bensley (1899) showed that in the fundus glands of the
cat and the dog the chief cells were of two classes, those of
the body and those of the neck of the gland tubule. The for-
mer '/ere characterized by the possession of a large number of
"zymogen" granules, which were distributed in varying ex-
tent throughout the luminal zone of each cell, and by a proto-
plasmic outer zone, distal from the lumen, which stained deeply
with nuclear dyes, and which also presented a fibrillar appear-
ance. These cells, he thought, were engaged in the secretion
of a ferment. Those of tiie neck of the tubules did not con-
tain, at any stage of digestion, zymogen in the form of gran-
ules, while their staining capacity indicated that they were
engaged in the se; retion of mucin. He held that the pyloric
gland cells of the cat and the dog were analogous to this latter
modification of the chief cells. The mucous neck cells of the
glands o" the fundus region of the fro^i's stomach together
with the cells of the bodies of the pyloric glands were, he
stated, the physiological and morphological homologues of this
type, with which they corresponded in both position and func-
tional changes as well as in staining properties.

From the foregoing it may be gathered that the structures
of the chief cells of the body of the fundus gland tubules and
the changes which they have been observed to undergo during
their various phases of activity indicate their main function
to be that of elaborating the pepsin of the gastric juice. The
function of the parietal cells, however, presents a more difficult
problem. Their structural detail will not permit of draw-
ing any sweeping conclusion. As Heidenhain a^rly pointed
out (1870) they were very large in comparison with the chief
cells, the cell body consisting of a very finely granulated proto-
plasm in marked contrast with the zones of coarsely granu-

CoLLiP: Formation of HyDRocHLORic Acid

lattHl protoplasm seen in the chief ceils. The only change ob-
served to take place during activity was in respect to their
bulk. They are larger during activity. The cell protoplasm
of the parietal cells stains very deeply with such dyes as eosin
and acid fuchsin, and their differentiation is thus an exceed-
ingly simple matter. They also occupy a position distal from
the lumen, but with which they are nevertheless in direct
communication by means of diverticula of the lumen extending
between the chief cells. The bodies of the parietal cells them-
selves are permeated also by a system of fine intracellular
canals which communicate with the diverticula. The fact that
the parietal cells are restricted in their distribution to the
({lands of the cardiac portion of liie stomach, taken in con-
junction with the physiological evidence that the secretion of
the pyloric glands is alkaline in reaction, seems to indicate that
these cells are in some manner associated with the acidity of
the secretion of the fundus elands. That they do not at all
exercise a pepsin-secreting fui.ction cannot, however, be main-
tained on histological evidence.

The parietal cells are undoubtedly a unique type in the
r Tinialian stomach. The question therefore arises: Are they
entirely a new development in this, the highest order of the
Vertebrata: or are they represented, or is their appearance
foreshadowed, in any of the lower Vertebrates? The idea
which has generally prevailed, as will be shown, has been that
the parietal cells are represented in the lower forms of Verte-
brates, and that they are simply a specialized and highly modi-
fied type in the Mammalia. Many authors, too, on such
grounds, have ascribed the peptic fu "*ion to the parietal cells.

The Gastric Glands of Birds. The stomach in birds con-
sists essentially of two parts, the glandular stomach or
"Driisenmagen" and the muscular stomach or "Muskelmagen"
with, in many cases, an intermediary zone between these two
regions. The muscular stomach has a chitinous lining which
is being constantly renewed by the secretion from the modified
mucous glands. The glands producing the acid and the pep-
sin of the gastric juice are entirely confined to the "Driisen-

1 i

"m

8

CoLLip : Formation op Hydrochloric Arm

magen" or "first stomach." This organ is manifested as ,,
dilation of the oesophagus for a short distance before its jim,
tion with the gizzard or muscular stomach. The wall <if tin
region is thickened, owing to the presence in it of the ospcciall-,
modifi'Hl and very much aggregated gastric tubules in the f(.iii
of compound glands. The opening of each of these comp.HiiKi
glands into the central lumen is marked by a papilla. When
the "first stomach" or proventriculus, as it is called, is laul
open the wall is seen to be dotted with these papillae. Th.
mucosa of the wall between the papillae is folded into slid't
mucous crypts.

Reaumur (1752) noted the presence of the papillae, whi!.
Molin (1850) described quite accurately the finer strudiut m
the proventriculus itself. Hasse (1866) stated that tho .scciv-
tion expressed from the compound glands was quite dtai
Cazin (1888) divided the cells forming the compound plaiuls
into two classes: the peptic cells, which formed the periplmal
tubules, and the mucous cells, which lined the wall of the col-
lecting channels. Klug (1893) described the peptic cells of thi
compound gland tub"les as being finely granular and haviiur
no restricting cell membrane. The nucleus he found to bi
more or less central in position, as is the case in the parietal
cells. He held that these cells, which he found to be all of om
type, secreted both the acid and the pepsin of the gastric juia ,
and that they were homologous with the parietal cells of the
Mammalian gastric tubules.

Only one type of glandular cell has, therefore, been recog-
nized in the peripheral gastric tubules of the compound glands
of the bird's proventriculus. This type may be compared with
the parietal cell of the Mammal, yet such a comparison can
only be of the most general character. The cell body itself in
its staining properties is more like the chief cell. But here
again, there can be found no broad basis of compari-
son. It subserves the same functions as both the parietal
and chief cells combined, and therefore one might expect that
points of resemblance with both these types would be found.
and indeed this seems to be the case.

CoLLiP: Formation op Hydrochloric Acid

The very slight amount of interstitial tissue which can be
Jomonstratcd between the compound gland tubules is note-
worthy, and as the Mammalian stomach, large lymph
nodules are of frequent occurrence on the walls of the secret-
ing tubules.

The (histric Glands of Reptiles. The stomach in the Rep-
tilia is usually very much elongated, but the pyloric and fundus
retfions are always capable of definition, microscopically at
oa-st (Oppel 1896). The glands of the fundus vary consider-
ably in size and structure. The crypts may be very deep and
lined by the typical neck cells of the mucus-elaborating variety,
or again, comparatively short but always showing the same
typo of neck cell. The body of the gland tubule is constituted
if one type of secreting cell. The pylorus gland cells resemble
tho neck cells of the fundus region.

Partsch (1877) showed that both the pyloric region of the
ftomach and the oesophagus gave extracts with a very low
pepsin content, while from the fundus region extracts of high
digestive power could be made.

The Gastric Glands of Amphibians. The body of the "Ljib-
diusen" or peptic gland tubules of the Amphibia contain but
one type ceil (Oppel 1896). The neck cells of the fundus re-
gion and the pyloric gland cells afford practically the same
comparison as their homologues in the Reptilia and Mammalia.
In Batrachians, such as the frog (Langley, 1881), the peptic
glands are not confined to the cardiac portion of the stomach
lor the oesophageal glands secrete pepsin, but not an acid
juice.

The Gastric Glands of Fishes. The peptic glands of the
order Pisces are formed of one type of cell only. The size of
the cells and the nature and extent of their granulation vary
in the different species. They are, generally speaking, of the
same type as their homologues in the Amphibian or Reptilian
stomach (Oppel, 1898).

Richet (1878) noted that the hydrochloric acid in gastric
juice of certain fishes was represented by a concentration of
from ten to fifteen grams per litre. The highest concentration

i

1 i

.ttJt^

10

CoLLip : Formation of Hydrochloric Acid

which has yet been observed in the Mammalian stomach wa-
0.58 per cent. > liosemann, 1907).

This review of the histological structure of the trastru
tubules of the lower Vertebrates suffices to show that in (oin
sidering either the acid or the pepsin-elaborating function <
these glands we shall, in all cases, with the exception of cciLim
Batrachia, have to deal with one type of cell only. Thea*- 1 . Hs
have the same functions as are performed by the parietal Mil
chief cells of the Mammals. An investigation which wmild
reveal the manner in which the hydrochloric acid of the gastric
juice of the lower Vertebrates is elaborated by the gland nils
of the gastric tubules should, therefore, throw some light ui)..ii,
and assist in the understanding of, the acid-forming function
of the Mammalian stomach.

2. The Data from the Application of Chemical Mi-tht,<l.^

The microchemical method was first applied by Claude
Bernard (1850) to det . nine the seat of origin of the hydm-
chloric acid. He injec ) nto the jugular vein of a rabbit a
solution of lactate of iron followed by one of potassium fono-
cyanide. He hoped thus to detect the place of origin (jf tht
acid as no precipitation of the Prussian blue could occur in the
circulatory system since the blood was alkaline, while such a
precipitation would take place in any tissue or fluid of the
body which had an acid rciv tion when the two salts reached
that ti.s8ue or fluid. After three-quarters of an hour the ani-
ftial was killed and the stomach immediately laid open. A de-
posit of Prussian blue was found on the surface of the mucosa,
which was especially marked in the region of the lesser curva-
ture. Microscopic examination, however, revealed no blue
other than on the surface of the mucosa. Bernard concluded,
therefore, that the acidity of the gastric juice was only mani-
fested after the secretion from the tubules had reached the sur-
lace and had become mixed with the fluids of the stomach.

Briicke (1859) was of the opinion that the hydrochloric
acid was produced free in the gastric tubules. He pressed
freshly cut sections of the proventriculus of a pigeon, actively

p

CoLUP: Formation or Hydrochloric Acid

11

digesting, between litmus paper. The free surface of the
mucosa proved to be strongly acid in reaction while the multi-
lobular glands imparted a neutral or only a very slightly acid
lint to the litmus. The mucosa of a rabbit's stomach was like-
wise found to have an acid reaction on tiie surface but to be
neutral in the deeper portions. Briicke assumed that, if a
'reeacid was liberated in the gastric tubules, an alkaline fluid
if corresponding strength must at the same time pass into
the blood and lymph systems. Thus he explained the non-
occurrence of an acid reaction in the tubules themselves.

Lcpine (1872) repeated Bernard's experiment and ob-
Uined similar results. He tried also other methods in attack-
ing the problem. He macerated in a solution of lactate of iron
the gastric mucosa of an animal which had previously been
injected with a solution of potassium ferrocyanide. Negative
results only were obtained by this method. While he was not
able to show that either type of cell was acid in reaction, he
nevertheless held that his failure did not entirely prectjde the
possibility that oiio variety of cell had the function of pre-
paring the acid if not of completely elaborating it.

Edinger (1882) introduced several new method? He used
tropacolin and phenolphthalein, but with these he obtained no
definite results. Noteworthy findings were obtained when con-
centrated neutral solutions of sodium alizarin were employed.
Neutral solutions of sodium alizarin are purple red in colour.
The addition of a drop of acid causes the precipitation of the
yellow colloidal alizarin. He found that the gastric mucosa
of rabbits and dogs was flecked with small areas which had a
yellow colour after he had injected into their blood circulation
a concentrated neutral solution of this substance. These yel-
low patches were separated from one another by broad red
violet zones. The yellow colour could, in sections, be traced
into the deeper part of the tissue, but no positive information
as to its precise locality was obtained owing to the faintness
of the colour in thin sections. This reaction did not obtain in
the mucosa of fasting dogs. The occurrence of the isolated
yellow patches in the wall of the mucosa of the supposedly

^

L

12

CoLLiP: Formation of Hydrochlchic Acid

active stomach. Edinger explained as due to only small ar.ji^
of the mucosa being in full secretary activity at any ono tmi,

Trinkicr (1H81) also used tropaeolin with negative nsiilt.
He concluded that neither chief nor parietal cells formed fr. .
hydrochloric acid.

Stintzing (1889), using Congo red, which in the pres.nr,
of a mineral acid changes from deep red to blue, obtained m
some parietal cells of the fundus glands of various aiiiiii;il>
blue granules of varying sizes. This he held as evident sup
porting the view that hydrochloric acid was elaborated by tin
parietal cells.

Sehrwald (1889) utilized the Prussian blue method m a
modified form. The fresh gastric mucosa was placed for a day
in a solution of lactate of iron, then washed and transfiind
to a solution of potassium ferrocyanide where it remained r.n
some time. After this treatment th.' parietal cells were fmind
to stand out clearly, owing to a large content of Prussian hlu'

Frankel (1891) showed, however, that results similar t'l
those of Sehrwald's could be obtained when alcohol-harde-n.l
tissue was used instead of Iresh material. He also repeated
Edinger's sodium alizarin experiment and confirmed his find-
ings. He pointed out that this method was not without objii-
tion, however, as the reaction could be brought about by th.
acid resulting from the dissociation '' the neutral salt. Frinkel
injected intravenously decolorized uiutions of acid fuchsin.
In the active stomach of the dog he found the whole suifaci
of the mucosa stained a brilliant red. Teased out tissue
showed both the chief and parietal cells of the fundus gland
tubules to be coloured intensely red. The cylindrical epi-
thelium and the interstitial tissue were not c 'oured. He
only obtained the colouring in patches on the surface of the
mucosa of the rabbit. He considered that his results proved
that the gastric tubules were acid in reaction, but did not think
that the formation of the acid could be ascribed to any ono
type of cell.

Gmelin (1902) held that an inter-relationship between the

CoLLiP: Formation of Hydrochloric Acid

13

parietal cells and the formation of the free hydrochloric acid
waa not yet proven.

Miss M. P. FitzGerald (1910) undertook, under Professor
Macallum's direction, to determine if the Prussian blue reac-
tion could not still be applied to reveal successfully the seat of
rigin of the hydrochloric acid of the gastric juice. She em-
;)ioyod potassium fcrrocyanide and the double citrate of iron
and ammonia which was substituted for the lactate of iron
used by earlier workers. She found that weak neutral solu-
luti«n8 of potassium fcrrocyanide and the citrate of iron and
ammonia could be mixed and allowed to stand without a trace
uf Prussian blue being precipitated. The addition thereto of
jodium dihydrogen phosphate, sodium hydrogen carbonate and
carbon dioxide did not cause a precipitation of the Prus.sian
blue to take place. The slightest trace of hydrochloric acid,
however, brought about an immediate precipitation. The
double citrate of iron and ammonia was found to be neutral in
reaction, and in this respect wholly unlike the lactate and
other iron salts previously used. Both Lepine (1872) and
Sehrwald (1889) had observed the acid reaction of iron salt.s
in solution, and the spontaneous formation of Prussian blue
when a solution of ferric lactate is mixed with one of potas-
sium fcrrocyanide. A balanced mixture of sodium or potas-
sium fcrrocyanide (preferably sodium fcrrocyanide, as it is
less toxic than the potassium compound) and the double citrate
of iron and ammonia, makes, therefore, an excellent reagent
for the detection, by microchemical means, of minute quanti-
ties of acid in the body.

Miss FitzGerald, working with rabbits, dogs and guinea
pigs, obtained a sories of varied results by this method. After
intravenous injections of quite small amounts of soluflon
formed of equal volumes of an aqueous solution of 2.25 per
cent, of ammonium ferric citrate, and of a 1.5 per cent, potas-
sium fcrrocyanide, there occurred in nearly all cases a deposit
of Prussian blue on the surface of the gastric mucosa, which
was usually limited to the region of the lesser curvature. In
a few instances the deposit of Prussian blue was not confined

h

i

14

CoLLiP: Formation or Hydrochumic Acid

to the surface of the mucoM, but wai found to be pre««nt nl*.)
in the crypt* and, at isolated points, in the upper two-thirds
of the lumina of some of the gland tubules. In examination i,>
such preparati' lis with the high power objectivoi the canaluuli
of the parietal cells and their connections with the gland luni. n
stood out clearly, in consequence of the deposit of Frussum
blue within them.

Apart from the positions just mentioned, Prussian blue
was also fjund in some adjacent lymph vessels, a few IjIukI
vessels, and in certain wandering cells and leucocytes betwtcti
the gland tubules. In no other situation was a deposit «i
Prussian blue observed, but the immersion of portions <n' tli,
various tissues in dilute hydrochloric acid caused the bin.
precipitate to be formed, showing that both salts essential l(,i
the reaction were quite uniformly distributed.

The occurrence of Prussian blue outside of the gustru
tubules in the blood and lymph vessels and in the connectivi
tissue spaces. Miss FitzGerald attributed as due. possibly, ti.
a certain toxic action of one or other of the injected salts on
the gland cells, causing the discharge of free acid from tht
parietal cell in an inward, as well as outward, direction. From
her results she drew the following conclusion: — "The occur-
rence of Prussian blue reaction in the canaliculi of the paiie-
tal cells affords conclusive evidence of the presence of fm
acid within these structures." Although having no very
definite evidence on the point, she was of the opinion that thf
hydrochloric acid was f'^-meJ free within the cytoplasm of the
parietal cell itself, from ./hence it diffused into the canaliculi.
She thought such a view to be more tenable than one which
supposed the hydrochloric acid to be formed free in the
canaliculi only, and the parietal cell body itself to be alkaline.

Corroborative evidence that the parietal cells were the seat
of the elaboration of the free hydrochloric acid of the gastrir
juice was also obtained by Miss FitzGerald. Greenw(K)d
(1885), whose work has been referred to earlier in this paper,
found that the parietal cells could be differentiated from the
chief cells after fresh gastric mucosa had been treated with a

CoLLiP: Formation or ilYDtocHLoRic Acid

lo

» lulion of iilver nitrate. Professor M«callum ( I9or>) showed
that the staining propfrties of silver nitrate solutions were due
prrtctically entirely to the formation of insoluble silver c«tm-
[xiunds in the tissue when it was penetrated by the nitrate
r.agent. These silver precipitates became subsequently tranH-
formed by the action of light into coloured reduction com-
pounds, and it was these that imparted the characteristic col-
uurs to sections which had been treated with the nitrate of
Sliver. The silver salts, which were thus precipitated in the
tissue, consisted almost wholly of chlorides, phosphates and
carbonates. He devised also a method for the localization of
tne chlorides micro-chemically. When a solution of silver
nitrate containing 1.6 per cent, of free nitric acid was utilized,
ht' .showed that, with the exception of taurine and creatine.
only the chlorides in the tissues would be precipitated. As
tiiurine and creatine are not present in the majority of the
tissues, or at least never in any appreciable amount, a specific
reajfent for the detection and localization of chlorides in any
tis.sues was thus made available.

Miss FitzCIerald placed in silver nitrate solutions and the
special chloride reagent, respectively, portions of the f:e.sh
Ifa-stric mucosa. The vos.-»els containing the solution.s were then
exposed to sunlight for some time. Portions of the tissue were
then teased out and mounted on slides in glycerine. She found
that I' parietal cells were clearly defined in both instances.
Th" amount of the reduced silver compounds ob-served in the
chief cells and the interstitial tissue was relatively very much
le.ss than that in the parietal cells. The reduction manifested
in the parietal cell was more marked when pure nitrate of sil-
ver solution was the reagent used. The abundance of chlor-
ides, phosphates and carbonates in the parietal cells, thus
shown, was put forward by Miss FitzGerald as evidence fur-
ther indicative of the acid-forming function of these cells.

Harvey and Bensley (1912) repeated much of Miss Fitz-
r.crald's work. They also introduced many now methods of
study. They did not think that Miss FitzGerald's results had
settled the question, and held that all results obtained by the

?

1

I

-:^*^

16

CoLur: Formation or llYORocHLORic Acid

PruMian blue mt-thod would h«ve to be ruled out of conmdi ra-
tion. They, thcmw'lvtii. obtained ri««ult» aimilar to Mimii hiU
Gerald's by this method, but they found, as well, Prusnian hlu,
in practically every tissue of the iKidy. They claimiKl. ih.rt
fore, that the Prussian blue, which was found in thi- jiLand
tubules and the canaliculi of the twrietal cells, had not n.t.s-
sarily been precipitatt'd there, but might have bei-n forni.ri
eliM'where, and then have been transported thither by mtiuis of
the bliMid and lymph streams. How this transport of Pru.HM«ti
blue through membranes and cells could obtain they did ii,,t
explain for even the "soluble" form of this compound d<Ms rii.t
penetrate these structures. Further, the solutions of siKlium
ferrocyanide and ferric ammonium citrate used by thmi
were of excetdingly greater roncentrotion than thimr /,«.,<
Hiiiiilarly by Mum FitzGeraM. The latter in her expirimtnt..
had injected only very small quantities of a "balanced" pn--
pnration, which she prepared by mixing equal parts of a 2.2"
per cent, solution of ammonium ferric citrate and 1.5 por nnt
solution of t>otassium ferrocyanide. Harvey and Hmsley umiI
however, at times twenty-five per cent, golutionn of animiiniiim
ferric citrate and ten per cent, solutiunn of Hodiuni (iri>^
cyanide, mixtures of which, without the addition of amj arnl,
ffive eopioftn precipitates of Priumian blue. These they in-
jected separately. They never f'-nnd Prussian blue in the
parietal cells without finding it also .n the blood vesseLn. while
in .some cases tRey found it in the blood vessels without findinu
it in the parietal cells.

Deposits of Prussian blue were obtained on the .surface of
the mucosa of the proventriculus of the fowl, and also in d*'-
prcasions leading to (ho compound glands of the .same. They
did not observe any in the cells or tTio lumina of these ghmds.
but found them in the adjacent blood vessels and lymph .spaces.
as well as elsewhere, especially in the endothelial cells of Kupf-
fer in the liver. In the turtle Prussian blue deposits were pm-
duced in the epithelial cells of the foveoiae and the n«'ck cells
of the gastric gland tubules to a certain extent and in th(> cells
of the bodies of these glands to a slight extent. The reaction

Coujp; Fo«MATioN Of IIyt)*(K HM)Ru- Acid 17

*.. obUinrd in th- connective Ummuv .nd bl.MKi ami lymnh
...4. « .n th.. .Ml. dog and fowl, and U .K.urr.^ MnuU ly n

I xiHTim^nU di^igmnl to .how the efTwt of mjurv .,r the
i^luv mur.««. of th.. pr.....n.-.. of „o,«.,n«. «„d of r.-irtcted
^-« supply on th.. PruMun blu.. reaction, kuv,. n.or.. or |..„
•■it.t.v.. r...ult«. Parietal cIIm ,n th.. r,.«,on of ,„j„rv ,„ ,h."
1..«and cat wer.- found to U. loaded with l>ru«.an blu... |u..
.ruling th.. bl.HKl supply of the nu.co..«, cau.ed. in aon... ,.«,e«
^l,vM to arue. and th.-He were found alway- to be cottt..d with
° u«.Hian blu...

llu .KTurrence of Pru««ian blue in many tiwuea. ,uch a«

"' T!: /.T"^' '"■"'■• ""'"""■ '"t«t*»«. etc.. proved, they

la.m.d that the ri-action probably took place without the hejn

fa., acid. They suggested that nomo .such factor. «« th.- with-

drawal from the blood stream of the ammonium ferric citrate

.mv inpidly than th,. Modium ferrm-yanid... owin^ to it. easier

Jil u.ibility or the involvement of the ammonium Halt in the

.K-tabolic procesae- of the tii.i,u..«. or the d-ath or injured

iUl.ty of some of the c..|l«. might furniah an ..xplanat.on of

t» widespread occurrence.

By th,. use of a numb«.r of aniline dyes, particularly of
yanamin bichloride, they endeavoured to determine the local-
zat.un of the acid formation in the gastric mucosa. In the
prmnce of an alkali this dye becomes red. while in neutral or
^nd .solutions the colour is blue. When portions of gastric
mucosa, removed from actively digesting stomachs, were
^im-d out in saline solutions of the dye the cannliculi in the
panetal cells stained red. the lumina of the glands were bluish
^ and the canals connecting the canaliculi and the lumina
vere intermediate in colour, while the secretion as it obtained
nthe foveolac and even the cylindrical cells lininr them took
■n a blue colour, all in contra.st with the results observed in the
^astnc mucosa of the restihg stomach which did not give these
feactions.

Th,y obtained somewhat similar results with Nile blue and
neutral red. The latter dye is red in neutral solution and

.

I

IM

COLUP: Fc«MATION or IIVUSncHLOtlC Arm

rrimaon in Mcid ■ulutiiin, but when nthi^r u rvmli'ntl alkaiin.
a yptlow l»n«i' !■ prifH»it«t«d. Whrn tmrtlutti «>f the rr»'»hi>
ri'n>i>v»'i] Kastrir niuriHwi. arttvply •fcri'tintf. v/vrv tiaitrri ..ut in
tt »tilutton of oiu> |wrt uf thin ilyv in ten thuuaaml of jMlttt-
iMilution th«» lannlti-uh of th»» |Mtru>Ul cvWn wt-rt- yi'llnw. th.
Illitniiular lumina red and the cunal* conniHttng wirt* of att ir
tcrntcdlatc fuli»ur.

Thiwc rinulta |i*d tht>ni tu ittnc-ludt* that thr hydrm-hlMrii
arid waa not frt'i* aa «udi m thi* ffantric tubulci, but wan IiImi-
attd in th«- foveola*'. and m thf fpithi'lial fi'lla of th<« nut fa. . ,■
thi> trantiK' mucoBa. Th<' |>aru'lal crlU and the citntftitH i.i
their canaliculi they held to b4> alkaline. The (mrietal reil<,
they were itu'ined to hold, elaboratwl a rom|H)Und, posiitiK ,i
chloride of an organic base, of protein charactir, whlih uw--'
riao to free hydrmhloric aeid when it dilfuaea into the fovt-o'ae
and upon the xurfare of the mucosa.

Hanmiett (lUl')) ha* criticiw'd the evidence whirl' h.il
been put forward by both Mm.* F'itztierald and HeiiHlcy ar.d
I'arvey in regard to the jtource of the hydrochloric acid ir il,'
.stoma' It He held that the renitona which led Uentlfy ami
Harvey to reject the resulta obtaimni by the une of the I'm*
sian blue eX|H>rlment were not justified.

Their critii'idma concerntnl. amongst other reaults. tin r-
constancy of the reaction itnelf in th,. stomach on all <H'ra'«i.in»i
and when it waa obtained, its reatriction to limited ar>.ii
within whtHi 'nly a few of the parietal cella reacted. !!an-
mett hold.i thai legional activity, decreased bIcKKl supply am!
the toxic effects of the injected salts could account for soiti.
of these results, and that the n- ,J-reacting cells amonirst thi
reacting ones could be considered as in the resting stntr. or
to have already discharged their acid and by the toxic acti.m if
the injected salts to have been prevented from fiiii
activity.

Their mo.st weighty objection was that the reaction i« Hi-
red in other organs than the stomach. Ife attributed thi^ p-
sult to the presence of an acid, such as lactic, in the tis^inv
especially in heart muscle when il is dead or dyintr, wh li

CottlP: FfWMATtON or IfVWKK HI.««H Ac

ID

19

...ultl fxpintn th«' iKTurrtfnct «r Pruulan blur m thu tiaaui*.
J, •Ui-rv...! by llArviy mtii lUtul.y. wht>n norn- wu^ f.mnd in-
i!<t<' thr bliHid vt*iM><«U of thia III gun.

n«mni<'tt nmintAinrd. rontrary to llnrvi-y Hnd lUnalry'ii
.- jitH And toncluai«)n» m Ihlii p«iJrit, that wh.'ri m.lutn.n* of
»M<lium fiTrocynriulf nnd f.rric ammonium citratf ar<- mix.d
with bloiKl or bl mhI a.nim i« t i7rr, l'ru»«ian blu»' do«t» not form.
ml hi? dm'* not tndtc«ti» thi> fonwntrntlon of thi> aolutioim hi'
iM.%'. It u. howi'viT. probttblo thnt hi- usod roncfntrBliorm of
■htitf inlta ni»t trrcatt'r thnn thoa.- ua*-*! by Mijta FttxCH-rnld,

iiammi'tt n rnatid llnrvf. utul Hi-naliy'ii i-xpi ' iriu-ntH with
vitnHmin. Thm dy«', na alrmdy ixplairutl, givia dutiiutivf
r.a<tiona for acid. nlkHlirii- nnd mutral aohitiona. With it*
**«• h.> got thr anmo rt'aults an thoy did, but h«' pointM out that
thi- i»arii»t«l ci-lla, if ihoy iM>cr«>t4' mid, ahould Im«. aa waa found
UiU- thf ciia«', nikalino in ri'nction, ami furth<«r, that in pun-a
if the fn-ah mucosa rimovid and Inatcd with thia dyo a»Tri-
tton cw« s nnd the distribution of the dyv in th«' atrudun-a
ikpi-nda on tho rolativi- vfl.Kity of diffuMJon of thi- dye nnd of
thf acid which i:i irn-ati-r than that of the dyr. Ah ihv rate of
liiffusion of tho arid is grwitfr it is only whi«n> the dy*- and acid
frwly mingk'. na for exampio, on tho aurfneo of tho muroaa
and in tho fovoolao, that tho arid roattion i.h doHnitiiy indi-
ratiHi, whoroaa in tho lumina of tho ulnnd tubuloa and in tho
can.iliculi connoctin»r tho lumina and tho jmrit tal colia thoro ia
no intoraction of and and dyo Iwcnusc tho dye lU -a not dilfuao
into thorn.

Tho rosulta. thoroforo. Hammott maintaina, do not contra-
lict Miss Fitztlorald'a obsorvationa and conclusi<.nH which ho
rifards as confirmed by his own and Harvey and Bonsley's
rpsults.

^- I

K^,:MAn^-^-^i.':^^^'^-

&M

20

CoLLiP : Formation of Hydrochloric Acid

II. Methods of Study

A great deal of the vork in microchemistry in the past hiis
been done under considerable flifliculty. The results had
nearly always to be interpreted as involving the possibility
that a slight shifting or an extending of the area, to whicii the
substance being localized was confined, had taken plan'.
This shifting could be brought about by diffusion and osniosi,-,.
These might possibly exert a certain acti>)n in the time hv-
tween the removal of the tissues from the body and the ptiu •
tration of the specific reagent used to the innermost parts d
the same. The nearer, therefore, one can approach conditions
under which the precipitating reagent is brought into ininn-
diate and intimate contact with all parts of a tissue in which
the various constituents are still in their normal relation.^, thi
greater accuracy in the localizing of any constituents will
thereby be attained.

The freezing microtome is indispensable for this putposc.
With it, from tissues excised immediately after the death of
the animal. sect'')ns can be prepared without any delay in
which there is practically no alteration in the distribution ol
the salts as they occur in the normal living structures. The
sections so prepared must, however, be transferred while
frozen to the reagent to be used on them, for if they aio
allowed to thaw changes will obtain which will tend to give
confusing results. To prevent this thawing of the sections
the sectioning knife and the air about the microtome must bf
kept at a temperature below 0° C. This was effected by enclos-
ing the microtome in a wooden box lined on the bottom and
sides with asbestos and covered by a thick sheet of plate ghiss.
into which box led two tubes, each terminating with a spray
nozzle from a drum cylinder of liquid carbon dioxide, one to
use in freezing the piece of tissue on the stage of the micro-

m

^{.^^'^MM^^h^im^Jt,

CoLLiP: Formation of Hydrochloric Acid

21

t.mc. he other provided with a rrovable joint connection, to
spray the knife as occasion required, and also to chill the air
To permit the microtome thus enclosed to be worked an open-
ing m each of two opposite side walls permitted the introduc-
tion of the hands of the operator. With the dish containing
the reagent to be used, and with a camel's hair brush chiHed
t.) the temperature of the air in the box it was possible to
transfer the sections made, while still frozen to the contents
of the dish. This reduced to an absolute minimum the risk of
the constituents, inorganic or organic, difrusi^g from the
structures in which they are held in the normal tissue.

For the localization of inorganic constituents in the tissues
thus sectioned special methods were employed. These and
•others which were also employed in this research will now be
described.

1. The meih„ds far the detertim, „f chlorides and phns-
plwles. The methods for studying the distribution of the
chlorides and the phosphates and carbonates will be considered
t'iRother. The special reagent for the former consisted of a
decinoi-mal solution of silver nitrate which contained 1.5 per
cent, free nitric acid, that foi' the latter consisted simply of a
dtvinormai solution of silver nitrate (Macallum. 190.'-i). These
two reagents, when used in conjunction, enable one to study
the distribution of. and the relative relations existing between.
the chlorides on the one hand, and the phosphates and carbon-
ates on the other, in any tissue.

The distribution of the chlorides can be definitely deter-
mined, while that of the phosphates and carbonates can be
quite accurately decided by the method of difference. Whether
the excess reduction, which is observed in the latter case, is
due entirely to phosphates or in part to carbonates cannot be
determined, for there is no microchemical reaction for the
latter alone. These two inorganic constituents of the tissub
are. however, probably nearly always associated one with tlie
other, and the results obtained with silver nitrate alone, when
allowance is made for the presence of chlorides, should, there-

22

CoLLiP: Formation of Hydrochloric Acid

fore, be held to indicate the presence of carbonates as wili u.s
phosphates.

The tissue (which consisted of typical portions of the
mucosa of various vertebrate types) was always removed li.in
the animal as quickly after it was killed as circumstaiios
would permit. The larger animals were always anaesthetizi ij,
the smaller ones pithed. In every case the stomach was iinnu -
diately exposed and the portion of the mucosa removed, lio/.. n
and sectioned after the method previously detailed. Wlim
either the decinornial solution of silver nitrate or the "chhuidi'
reagent" was used, the sections wore allowed to remain in a
dark cupboard for a few hours in the respective reagent. '•■' u<
sections were then removed from the silver solutions by mtans
of largo carefully cleaned glass rods and transferred to di>hi>
containing distilled water. From this they were taken up on
slides which were then drained, a drop of 50 per cent, glyitr-
ine was applied to each section and a cover slip placed over it
The mounted preparations were then placed in bright sun-
light for a few hours. This completed the process. Tlie un-
parations were made permanent by luting the edges of the
cover slips with Canada balsam.

2. The method for the detection of phosphates alone. The
nitric-molybdatc reagent introduced by Professor Macallum
(1898) for the localization of phosphorus, chiefly that organ-
ically combined in animal and vegetable tissues, can also bi
utilized for determining the distribution of phosphates, with
more or less success, if applied in a special manner. This it-
agent is made by dissolving one part of pure molybdic acid in
four parts of ammonia and then adding this solution slowly to
fifteen parts of nitric acid of specific gravity 1.2. The pro-
longed action of the nitric acid will convert part of the organ-
ically combined phosphorus into the ortho-phosphate, and this
in the presence of the reagent is precipitated as ammonium
phospho-molybdate, which is yellow in colour, but becomes
dark green when it is "reduced" by treatment with a 1 per
cent, solution of phenylhydrazine hydrochloride. If, however,
the sections are treated with the nitric-molybdate reagent for

raMuajaMjik .^^

*m:

Collip: Formation of Hydrochloric Acid

23

a period of 5 to 10 minutes only, and at a temperature of
about 40^ C, only the inorKanic phosphate present will be pre-
cipitated. Further treatment consists then in the i eduction
il this precipitate by a one per cent, solution of phe lylhydra-
ziiie hydrochloride. This re<iuires but a few minut..s. " The
actions are then washed and mounted on slides in .jO per cent,
glycerine. The distribution in the section of the dark Rreen
•ivduced" compound indicates the distribution therein of phos-
phate salts.

:'). The nu'thnd for the detection of potansium. The method
lor studying the distribution of potassium embodies the use
"f the cobalt hexanitrite reagent (Macallum. 19< j. This re-
iigent is made by dissolving 20 grams of cobalt nitrite and .35
jranis of sodium nitrite in 75 c.c. of dilute acetic acid (10 c.c.
Hi' placial acetic diluted with distilled water to 75 c.c). The
solution of filtered and is then made up to 100 c.c. with distilled
water. Sections are treated with this reagent for but a few
minutes. The potassium is precipitated as the triple salt,
cdbalt sodium potassium nitrite, Co(XO,) ,3(KNa)N0.
-nH,0, n being either 1, 2, or 3 (Gilbert," 1898). This
salt is insoluble in ice-cold water which is, therefore, used in
washing the sections until they are quite free even of traces of
the reagent. They are then mounted on slides in a mixture
consisting of equal parts of ammonium sulphide and 50 per
cent, glycerine. The distribution of potassium corresponds
with that of the black sulphide of cobalt which results from
the action of the ammonium sulphide on the triple salt wher-
ever it obtains in the preparations.

I. The '•Prmsian blue" method. Small quantities of a
balanced" mixture of sodium ferrocyanide and ammonium
ferric citrate, made by adding 10 parts by volume of a 3 per
cent, solution of the former to 7 parts of a 4 per cent, solu-
tion of the latter, were allowed to flow slowly into a vein from
a burette. The solutions were always freshly prepared, and
when injected were tepid. After the injection and an inter-
val which varied in different cases, the animal was killed.
Small portions of the various organs were removed, fixed in

I

.4JF^

tfh

.if' *"^*

24

CoLLiP: Formation of Hydrochloric Acid

absolute alcohol for a period of at least 24 hours, then pas.std
through chloroform in the usual way to hard parafTin and
sections of them of 5-10 ^ in thickness were then made, whii h
were slightly stained with eosin and mounted in Canada
balsam.

Soluble Prussian blue was also injected into some animals
intravenously. A one per cent, solution of this substance was
employed. The method of procedure following the inji'cti.jn
was exactly the same as that just detailed for the 'Prussian
blue" experiments.

5. Polychrome B as an acid indicator. This dye which
gives a reddish-brown colour when added to neutral or alka-
line aqueous solutions, is precipitated as a violet-coloured
compound when a solution of it is rendered acid in the slisrht-
est degree. Acid salts such as sodium di-hydrogen phosphatn.
when added to a neutral solution of thfs dye, will also effect its
precipitation. It was injected intravenously into some am
mals. A 0.7 per cent, solution was used and only small quan-
tities were given. In large amounts it proved to be very toxick
The precipitate is somewhat soluble in water, alcohol, and ti>
a certain extent, also, in glycerine, so that no permanent pre-
parations of tissue, from animals treated thus with this solu-
tion, can be made. Sections made by the freezing method, of
portions of the freshly removed gastric mucosa of the ani-
mals were at once mounted direct from the sectioning knife.
in glycerine, and immediately examined under the microscope.

6. Histological fixation and staining methods. In these,
saturated , solutions of mercuric chloride in water or, as in
Bensley's modification of Foa's blood-fixing fluid, in alcohol
with equal volume of 3 per cent, aqueous solution of potassium
bichromate were used. Preparations fixed with the former
were hardened in it for 24 hours, those made with the latter
were kept in it for one to two hours. The dehydration and the
sectioning, staining and mounting were carried out in the
usual way. Absolute alcohol alone was also used as a fixative.

Lil^'.J»«AJey

CoLLiP: Formation of Hydrochloric Acid
III. Results

The results obtained with the different classes of the Ver-
tibrata studied will now be described, in detail.

MumtnaLi

The chief Mammalian types studied were the dog, cat and
rabbit. Some difficulty was experienced in keeping animals in
a iit-rmal condition, as well as in getting them into the particu-
lar state desired for any definite experiment. Pavlov's
■psychic factor" in digestion was noticeable in several cats.
Tho fear which they showed when they were in the same room
with dogs seemed to inhibit quite effectively their gastric se-
dition. Rabbits are often very "isatisfactory to use as it is
uniy rarely that their stomachs uic found empty.

The Resting Mamthalian Sttnnnch
The applicatio i of the method for chlorides demonstrates
only an almost m ^'ligible quantity of the same in the gastric
tubules of the fui dus region of the resting stomach. In both
the rhief and par etal cells chlorides are present in traces only.
The interstitial tissue, on the other hand, is very rich in these,
and there is also a heavy condensatiim of such salts abont th«
gland tubules. The lumina of the tubules may contain a slight
amount.

Sections, treated after the method for phosphates and car-
bonates, stand in marked contrast with those prepared to show
the localization of the chloride. A distribution of these salts,
similar to that of the chlorides, manifests itself in the con-
nective tissue, cells ana fibres, and about the outer border of
the tubules, as well as to a slight extent in the lumina. The
parietal cells, however, stand out in a remarkable manner.
They are seen to contain an exceedingly voluminous d^osit
r>f reduced silver compounds, clearly indicating a very great
content of phosphates and carbonates on the part of the parie-
tal celLi. Practically every parietal cell seems to be picked out
in this typical manner, while the chief cells exhibit only
fliijhtly more "eduction than they ha<l in those sections which

I

^6

Couir: Formation OF Hydrochloric Acid

htul hen treated for rhloridfn alone. The inorganic salt con-
stitut'nta of tho parietal cells are confined entirely to ihf
cytoplasm of the same, as in no instance has any such salt ben
observed to be present in their nuclei.

The Actire Mammalioi' Stumarh

In the fundus region of the actively secreting mucosa thi iv
is a condensation of the chlorides on ^he external faces of thi
tuuules and in the interstitial tissue, similar to that indiviitd!
above, as well as an appreciable deposit in the lumina of Uw
tubules. The chief cells of these are quite free from chlorides
as they are in the resting condition. On the other ho ml thi
rarietal relUs, in arfrw more or lesn dincontiniious, are tvliv-
ordinarily rich in chlorides and the canalit connectinu tins,
nils u-ith the lumen i)i each tubule are clearly and sliiiii)hi
outlined by the chloride reaction developed in them. Outside'
these areas, however, some groups of parietiil cells, more ( i'
less limited in number, may give no more marked a roactiuii
for chlorides than do those in the glands in the inactive condi-
tion. It is very probable that the tubules in which this (imii-
red had not begun to secrete.

The use of the method to detect the occurrence of phos-
phates and carbonates reveals the same concentration of those
salts in the parietal cells that obtains in them during then-
resting stage.

The occurrence of the reduced silver compounds in the
parietal cells is go".erally in the form of very fine granules
closely approximated to one another and distributed through-
out the cytoplasm of the cells. It is, however, to be noted that.
in the immediate vicinity of the nuclear membrane and to a
much less extent within the cytoplasm immediately adjacent
to the inner or luminal border of such cells, there is a greater
condensation of these granules than elsewhere in the cyto-
plasm. This peri-nuclear condensation is, in a way, very re-
markable, and was observed to be present whether the salts
present were those of chlorides alone, or of these and the phos-
phates and carbonates. So marked is this condensation, at

Collip: Formation op Hydrochloric Acid

•27

tnu.s. that the granules constitutitiR it have the appearance
1 forming a folid envdope of reduced silver compounds en-
Jusing the nucleus.

The application of the cobalt hexanitrite method for
potassium demonstrates that this element is present only in
Maces in the gastric mucosa, while the parietal ceils app»ar
to be (juite free from it. This would imUcntv that the ini,r-
',!'"(<■ xaltn which are present in the imrielal cells are tint those
7 iiotaiisiuM, but ctmsist probably veru laryelu of compoioKli
■ f .^odiion.

The use of the nitric-molybdate reagent on the frozen sec-
tions showed that phosphates were abundant in the parietal

(I'lls.

A rabbit, which had been kept from food for two days, was
iillowed to partake of a hearty meal of oats. One-half hour
later 20 c.c. of a 0.7 i)er cent, solution of Polychrome B was
injected in the auricular vein. No toxic .symptoms were mani-
fcstoii by the animal. Ore and a half hours after the injection
had been given the animal was kiHod. Sections prepared after
the method earlier set forth showed on examination a diffuse
blue colour in the parietal cells. This was particularly in evi-
dence in the portion of the cytoplasm adjacent to the inner
•>r luminal border of each cell. The parietal cells of the upper
third of the bodies of the ga.stric tubules of the fundus region
were in this manner specially demonstrated. It is probable
that this reaction is due to the presence in these cells of the
acid phosphate of sodium (NaH, POJ which, like a free acid,
Rive a blue violet precipitate with the reddish brown solution
'if this dye.

Neutral red .vas also used. Frozen sections of the mucosa
of active .stomachs were droppod into dilute solutions
(l.KtOOO) of this dye. They were mounted after a few min-
utes on slides in 50 per cent, glycerine. Sections were also
placed in dry slides and a drop of neutral red applied. Five
minutes were allowed for steining to take place, then a drop
"f glycerine was added and a cover slip placed over the sec-
tion. Both of these methods yielded similar results. The re-

I

I

2M

COUIP: FORMATIUN OK (hOROCHLOItIC ACID

•ulU were, however. t<K) jndoAnite to draw any siKtiflr <n(i.
elusion. The parieUl cells wen not observed to take on th.
alkoJine shade of the dye. nor did the canaliculi. A diffu.i. r. d
staining throughout the section was rather the conditi.,,
observed.

Rinh

As the proventriculus of birds C(>ntain.s the glands whidi
correspond with those which are to be found in the f^lndu^
region of the gastric mucosa of the Mammal, prepaniti, ns
from this organ in the hen were used in obtaining the results
which arc now to be detailed. The hen was chosen to npi.-
sent this class of Vertebrates because in it the organ is typimi
of the Avian proventriculus. and al.so because the animal could
be made to undergo the conditions required in the experinu nt»
without difficulty.

The Resting Prorentrieuliin of tkf Hen

The chloridcH are in yreat abundance condenHed in tin «-
tcrtuhular tisane of the compound ylunds and around thi
tubules themselves. The lumina of the tubules are not muih
in evidence in preparations made to show the distribution of
the chlorides. The gland cells are for the most part quite fif.
from chlorides, but a few cells manifest a slight chloride nm-
tent which, however, is quite uniformly distributed through-
out their cytoplasm. Occasionally a small restricted area in
sections of the organ can be found in the cells of which th.n
is a slight condensation of chlorides. The latter are chiefly
localized in that portion of the cytoplasm adjacent to the
luminal border and the neighbouring margins of the cell. As
will be indicated later, these restricted areas constitute -small
groups of actively secreting gland cells.

The results of the use of the method for demonstrating
the phosphates and carbonates show that these are abuiidant
in the connective tissue and also about the tubules in the form
of lymph condensations, but the portion of the cytopuu-on of
each gland cell adjacent to the lumen, equivalent appraxi-
viately to one-fhird of its volume, manifests a remarkabk

COLLIP: Formation (.F »vi)iiocin,oRic Ant) 29

at, ration wUk th-nr naUn. So heavy in the dt.p.mit in this
-.KM.n that undtT a loniparatively l„w magnification thf
m\\n& appear to be bounded by a 8olid black •'reduced" silver
d.'p.)sit of irregular outline. Kxternal to th.si. .lepusits and
i!stal to the lumen, the cytoplasm of each cell is free of any
i.posit. but this clear zone is, in its turn, bounded externally
>i,v the rt^dish bro.n deposit due to a "reduced" silver deposit
in the lymph condensation of the various salts about the
:ubulc8. Ah i„ the cohi- „f the imrietal nils „f th, nutiny
MiinimalUin Hto„iach thin romlitim, m remarkable on armiint
■I itH uniformity of ornirrenre in all the gUinil fithnhs of the
renting proventrirnlitti.

The Actire Proventrirnlux

The use of the method for chlorides demonstrates the
usual condeiisation around and between the tubules, but the
cells, unlike those of the rostinK gland, are well defined. Then
knre a inarned cond^nnation of rhlori/lex within them which is
nlmnst entirely confined to a narrow zone in the cytoplmm of
each cell adjacent to i/,s luminal border, and to the contiguous
Mugimt extending outwards towards the ba-ial borders.

Treatment with the method for phosphates and carbonates
W>" indicates a similar distribution of these salts. Under low
P'>wer magnification they appear to be condensed along the
luminal borders of the gland cells and from this zone of con-
densation deposits appear to extend between the gland cells
towards their basal borders. Under the oil-immeraion objec-
'!ve. however, the heavy black deposit is seen to be confined
intirely to the cells themselves, localized within each cell in a
sharp and clear-cut manner, as already indicated.

/" the active proventriculus, therefore, the chlorides, phos-
I'lidtes and carbonates are fon-td to have a like distrU)ution in
the cytopUxsm of the gland ci n. The phosphates and carbon-
■itcs are, of course, considerably in excess of the chlorides and
Kcur in the position just dtscribcd quite uniformly in the
cells throughout the glands. The distribution of chlorides in

4

so

CoLLiP: Formation ur Hyoroc it LoRic Acio

Iht'W typical |H>iiitiun», whilo quite fxtt'tulv», in nut. h«w.\.r
an uniform a» that of the phoMphatcH and carlMtnatci.

The I'ruMiari blue expenniotit. the complete methiHl ,,
procedure for which Iimm bvvn previounly uutlined, wan <.ir-
ried out on h»nit quite Huccenafully. The quantity of "Imi
*nced" mixture injected v«ri»'d in amount between ten ahl
twenty cubic centinietren. The brachial vein wan found I.. .»
the moMt serviceable for injectionit. A sliKht t«>xic elfeci vs i
usually manifested, but thin did not appreciably allect the u.:
nialH an they would always continue to jM'ck up srrain otln, I
to them after the injection had been Kiven. The inje< t.. i
waa, in all cixhvs, made three hours alter the fowl had i.
One hour after the injection had been Riven the proventru u i,
was removed, opened, carefully examined and small poiti..,-
removed and tlxed in absolute alcohol. The contents of tl..
muscular sttimach. as well as the surface of the mucosa <■( th
proventriculus, were in all cases found to be acid in reacti. n
A deposit of Prussian blue was always observed on th,
mucosa of the wall of the proventriculus. In some instan.i^
this deposit wa.s very heavy and appeared to cover the eiitin
surface of th« mucosa throuknout the length of this orjran.
In other cases the Prussian blue precipitate was conliiiKl
chielly to the mouths of the papillae while the mucosa betw. i u
these showed a light deposit of varying intensity. Mun-
scopically, also, Prussian blue could be occasionally seen in tin
large ducts of the compound glands when free hand acro.s.s sn-
ticms were made of the proventriculus. Microscopic exaniina
tion of alcohol-hardened tissue, sections by the parallin methyl
and slightly counter-stained with eosin, showed that Prussian
blue was present in the mucosa immediately adjacent tn tlic
heavy surface deposit. It occurred in slight traces in tlu
interstitial tissue of the tubules of the compound glands. It
was also present in the lumina of the.se tubules as well as m
the large collecting tubules and the necks and mouths of ex* n-
tory duct opening immediately by the papillae. The gland cells
of the tubules never manifested a deposit of Prussian l»lui
within them. As just stated, however, a deposit was to l>r

COU.IP: F(«MATION or llYDiOC'HL(«l« ACID

at

i.«rvi.tl at tini.'s in th« luminA of thi- tubuU'i.. In »u«h caM^a
It wtt» priiionl fithi-r ah a iiulid pluir. pruitually fllliriK th*
liiiiu-n, or the ,t nceurrtd „i the form „f a fi,„ aifnthtr ,,rr.

,',tntf uhiek f,.lluued thr cmlour of th,' Inminal wall
VVh.thfr in this laltiT ca«.. thi- dirKwiit was adunlly within
th.. Rland ii'll« or condirimtl only on tht- outnide wan dilllcull
to ii»>tfrmin«'.

The Pruiwian hlu«> was not oonflm-d to thi» prov.ntriruliw,
iu.«i.ver. Vi-ry HliKht trar»H „i it witp found in the conn.-ci
tiv.' tixsui- of both thi' paninuw and th«> intrntinal tract. Utart.
iu!i« and iplifu wi-rt' fr.-o from it an a rule, whil.- in the liver
a was «>bHt'rved in the endothelial celU of KupH. r.

In order to determine how 1'riisj.ian blue, which niijrht be

it-iulatiuK in the bl(H>d stream, would be Anally diJ^poned of, I I
> I. of solublo Prussian blue were allowed to Kra' itate mto
the brachial vein of a fi)wl. No untoward symptoms were
niiiiiifested by the animal. After 10 minutes had i-lapsed the

inimal was anaesthetized and portions of the various tissues
tt.re immediately placed in absolute alcohol. A small cpiantity

: a lijrht blue fluid was present in the cloaca, otherwise no
lilue was seen macroscopically. Microscopic preparations (,f
the various tissues prepared accordiiiR to the meth(Kl earlier
iiulitated, demonstrated a certain amount of Prussian blue to
fK- present in the connective tissue, the blood-vessels and the
lymph channels. The only place where it was observed to be
.It all abundant was in the endothelial cells of Kupffer of the
liwr. The proventriculus was no exception to the general
statement just made. Prussian blue was observed in its in-
ttrstitial tissue as in other organs. It was very worthy of
note, however, that there was no deposit of FVussian blue on
the surface of the mucosa nor in any of the gland cells, nor was
Mi< h a deposit observed in the lumina of any of the tubules.

HintoUiqieal Findinys

The proventriculus of a fasting fowl was fixed in Bensley'.s
fluid and preparations finally made according to Method «.
The luminal portion of each gland cell in these preparations

* f i

n

^

CutXir: KUCMATION ur HYOftCX'liLiMIIC Acio

wa« fiMtnd to Ih> markMlty MUinMl with rtiain. while thf otitfr
ur (mmI purttuo of i^ch tell wiu not no aUlntKl. iind th«-y pn-
svnM N MirilUir •pp«iiranc-c nnd m dirfuM' hm-miituxyhn aUm
In thf iictivi* pruwntrirulua then* wn* no Buth dlff<«ritituiii>ii
with ttmin in the rytopl«iim which wsn utAlncd uiuformU
thn>uirhout. Th«* nucU'UD. which, howovpr, did not atain «ith
iHwin, WWII t)lM«>t-v«-d to li«' richer in chronmtin nuit«>riMl thiin m
the rating colU. The cellM thi*mM*lv«a wcrt* ()«<rhA(M HliirhH.
itmaller In the active thnn in the resting gland.

When other (bcativcH were employed »uch di(Teri>ntiii(i<>n>
an theMr could not Ix* diiitinguliih'.>d »n readily.

The Turtl,'

Thv condition manifeated in the tubulea of the fumiu*
glanda of the stomach of the turtle ia very aimilar to that in th>'
fowl. In ttu iilami eelU in the rf/itin ■ rmiditioH the cytnftUii'it.
of the half of rarh cell adjacent to the lumen in very rich <«
phimphateH ami carboiiatj'ii, not a trace of which ia to Iw found
in the remainder of the cytoplaam. The Iwundariea of the cilN
cnn be diatinguiahed only with difHculty. The gland tubulin
are for the miMt part quite free from chloiidea, but, as in the
hen, a few scattered cella contain them. Thtme aatta art- uni-
formly diatributed in the cytoplasm of auch cells.

The reaulta of the uae of the method for demimatratinjr thi
phoaphatea and carbonates in like preparations from th*> !«t<'ni.
ach in the active stage nhnw that theiie are present in the rdh
uf the gland tubules, but more sharply defined in the porto-n
nf the cytoplaxm of each cell immediately adjacent to the hinim
than w the cant in the celln of the resting glands. The rciirti';-
of chlorid4ig shows a similar distribution. The conden.><atiim
of all these salta in the lymph about the tubules and in th«' in-
terstitial tissue can be learly demonstrated in all cusci*.

The Frog

The distribution of the rhloriil^s, phosphates and raihi.n-
ates in the gland tubules of the fundus regi^>n of the ii-i'ii'<
stomach (the oryntic glaiuis of Langlev. 1881). co>rcsii'.,iil-.

(•UI.UI-. F.HiMATioN or Uumn m.,mi Aau .t.i

i^ .. »r»(, rl.M^fn H,lk thnt »6-f n r,/ ,„ i/,. t. . , „

■*.n.-.. of <hl..rici.-H fr„rn th.'n.. r..|U ».,.h i '"""'•"''^'••
^ '-i ''.V IVor...... M«ca.Ln (,!;,;','* '"' ''"'"' "^•■^'""•"^

yt. AiHUt from th,. honnnl r.„„U„.„t,.,„ the ulaJcIN

IV. Gkneral Discussion

' vJ^'mTrn^r' "T'"''"""*' "' '''^' '•"■■'"'^^ "^ ^»'^' '"ventilation
. v.n in tlu. pri-cedinK pajri's may Ik- now di-HiuHsi'd

Th.-rc never was any doubt about the particination of thn

"ior,rle.,. onpecially that of «.Hlium. in the reacl^^on n ?h ' «-

nc .mKo«a by which the hydrochloric acid of the gastric .r.

re! of th? M """^ ^'rT^' ""^ '^ '"'^•^' ««TordinKly be the
oes„Kod„, ethe nature of the reaction which produced the
"1UCOS.I ,n which the reaction occurs.

i

r^

34

CoLLiP: Formation ok Hydrochloric Acid

It is pt-rlincnt here to refer to some views as to the rliaj-
jutor of the reaction to which the findings of this investigalio i
give special interest.

Maly (1871) obtained free hydrochloric acid by acting 'ii
lactic acid with sodium chloride. He was of the opinion tliai
it was in same such manner as this that the hydrochloric a( ; I
of the gastric juice was formed. Later, however, he chanutd
his views on this point (1878). This, he finally held, wa,^ i^.i
the normal nethod for the formation of the hydrochloric ;u id.
It might h>- the method to a certain extent in dyspeptics, hut
he held tli. in the normal stomach the hydrochloric aciii i>
liberated tnrough the mass action between sodium di-hydr -
gen phosphate and sodium chloride in the cells of thi- ;jra>tii
mucosa. The blood, though alkaline, contained acid salts ;ni i
so such a reaction would be possible. The free acid wniikl
have to be swept away as fast as it was liberated, whili 'h>
supply of acid phosphate would be kept up by the acti(ni ^ i
free carbon dioxide on the basic salt formed along with id.
hydrochloric acid. Maly's theory may, therefore, be expi r>s, d
by the two equations :

1. NaH.PO,+NaCl Na,HPO,+HCl.

2. Na,HPO,+CO,+ H,0 NaH,PO,-^NaHCO,.

He noted also, that the urine of dogs during gastric acti\ity
was strongly alkaline. He thought that the degree of alk .im-
ity of the blood was normally kept constant, and so wlui!, :;
any tissue, free acid was being elaborated from the blood, th
acid secretion of the kidneys would necessarily lag behind.

Von Noorden (1886) estimated the amount of the carbon
dioxide in the arterial blood before and after feeding. He
c .Id not detect any regular increase in the alkalinity ot th«
blood by this method. He concluded that the secretion of
acid in the stomach had nothing to do with the alkaline tide
observed in the secretions from the kidney during the period
of gastric digestion.

Bunge (1889) held that the hydrochloric acid was formed
by the interaction of the parietal cells according to the method
indicated by Maly. Carbon dioxide, he said, would be avail-

CoLLiP: Formation of Hydrochloric Acid

;jr>

iLlt' free, by the action of a ferment,
•rifiinic combination.-'

or by the oxidation of

The results obtained from the study of the distribution of
n chlondes. phosphates and carbonates in the pastra-

K d fferent stages of the.r activity -ustifies the adoption of

•Ki n th:7 ""' ';>'^" ""'^' •"' "••'^'" "f '^^ hydr'ochlo i
■u i. In the Mammahan stomach, where two types of gland

''?. ">.the bodies of the gland tubules of the fifdus

:•.', on. the d.stnbution of the various salts shows that th

acid-elaboratmg function belongs to the parietal cells Th'

. .sonce of chlorides in these during thei. resting age ad

he.r presence m them while in the active stage fs of unn s-

•al^ble s.gn.ficance in this respect. These cells, during mt

..nd act.v.ty. appear to possess the ability to store up and re

•nn phosphates and. very possibly, carbonates. Chl,"rid Is

uwever. are not taken up by the parietal cells until thev are'

- go mto acfon." The latte.- are then admitted and con-

.nuo to be admitted as long as the gland cells are secret ng

There ,s probably at the same time a high carbon dioxide con

. ntrat.on m the cell or its immediate vicinitv to ensure an

iTvdroch7 ' "' '•'; '"' '''"'^P'^^" '' '' ""t "-babio that
^ lid.och lone acid occuis in any quantity in the parietal
themselves, but diffuses as quickly as it is formed fnto the
m lacellular canahculi from whence it passes bv the diverti-
cula of the lumen into the latter. '

The findings from the method embodying the use of Poly-
home B indicate that the parietal cells contain, during their
ac hve state, either free acid or acid salts. The absence of the

rl'f..K'f' J '^'P''''* "^ P'^^^^i^" blue in parietal cells
^htn a balanced mixture of solutions of sodium ferrocyanide

Irr^T ^T'" ''*'**' '' '"J'^t^d '"to the circulation
makes it certain that free acid does not occur in their cyto-
?Tl 'S' therefore, very probab'e that the reaction with
Po chrome B was due to sodium di-hydrogen phosphate, the
P esence of which, as postulated in Maly's theory of the origin
^t the hydrochloric acid of the gastric juice

I

i

36

CoLLiP: Formation ok Hydrochloric Acid

In the light of all the evidence which has been con.sidci.d,
Miss FitzGcrald's findings are confirmed, and her intcriiivu-
tion of thoni must be accepted. Attempts to minimize th. :;
significance, it seems, have not been fully justified.

In the fowl we saw the samo tendency on the part of th,
gland cells to store up phosphates and carbonates as is mani-
fested in the Mammalian parietal cells. The typical condensa-
tions of chlorides, phosphates and carbonates in the pi tp.d
cells of the gastric tubules of the active proventriculus, which
have been described, and the absence, for the most part. . t
chlorides from these cells during the resting state would. tiihiHi
in conjunction with the results of the injection of the "bal-
anced" mixture of solutions of sodium ferrocyanide and
ammonium ferric citrate, indicate that the hydrochloric acid .,i
the gastric juice of the hen is formed by the glandular (vILs
of the tubules of the compound glands, and that it is liberated
either immediately within the cell border or at the cell margin
and diffuses into the gland lumen. The cytoplasm of the.«e eel-
possesses the power, when stimulated to activity, to con '(n
trate large quantities of chlorides, phosphates and carb- le.^
in the zones which are adjacent to the luminal border and the
contiguous sides. Such facts would indicate that the hydro-
chloric acid is formed as a result of the mass action between
these salts.

In considering the significance of the results of the injec-
tion, in the hen, of the "balanced" mixture of solutions of
potassium ferrocyanide and ammonium ferric citrate it is well
to remember the relatively great amount of hydrochloric acid
present on the surface of the mucosa in the active proventri-
culus. A certain amount of this may, under special conditions,
undergo reabsorption by the mucosa. This would account for
the occurrence of Prussian blue external to the cells secreting
the acid. The same observation may be applied to explain
irregularities observed in the occurrence of Prussian blue in
other orders of the Vertebrata. Further, the presence of lac-
tic acid, e.g., in striated muscle and the toxic action of these
salts in certain concentrations, which may alter the direction

"J-

Collip: Formation of Hydrochloric

f the current of the secreted acid,

Acid

M7

y very possibly account
K.r otner peculiarities in the di...nbution of the Prussian blue
observed. The occurrence, also, of the reaction on the endo-
thelial cells of Kupffer is intelligible. The.se cells, as Professor
Macaflum (1895) found, absorb in an unusual decree the salts
; iron in the blood stream i)assing them and, if they absorb
i^ntassium ferrocyanide as readily, Prussian blue must by mass
anion form in them as it does when concentrated st)lutions of
iiotassium ferrocyanide and ammonium ferric citrate are
:nixed in a test-tube.

The production of Prussian blue in tissues elsowheix' than
in those of the gastric mucosa without the participation of an
..cid in its formation, therefore, does not destroy the value of
the "balanced" mixture of potassium ferrocyanide and
ammonium ferric citrate, when appropriately used to detor-
mine in what cells of the peptic tubules hydrochloric acid is
formed.

The absence of chlorides in the parietal cells at points or in
restricted zones in the actively .secreting stomach, while sur-
charged as such cells are with phosphates and carbonates, in-
dicates only that not all the gastric glands are in full secret-
ing activity at any one time. This is quite in accord with the
(ibservations of Brown (1912) on the excretion of potassium
salts by the kidney, who observed that while groups of the
renal tubules were engaged in this excretion other groups here
and there in their immediate neighbourhood appeared wholly
inactive in this respect. The assumption, therefore, that all
the cells of a glandular organ when it is secreting are in action
at the same time, is not justified by the facts so far ascertained.

A brief survey of the results obtained in the turtle and
the frog makes it clear that these are in line with those ob-
tained from the mammal and the fowl. The fundus gland cells
of the turtle and the cells of the oxyntic glands of the frog
contain, at all times, great quantities of phosphates and, very
possibly also, carbonates. Chlorides were comparatively rare
during the resting periods with the exception of a few odd
ceil.s in which they were manifested to a certain extent. Dur-

U

38

COLLiP: Formation of Hydrochloric Acid

iriK activity tho condonsations of tho two (or three) <l;,.s.. -
of salts seen to occur in the gastric glands cells with (h ,,
actcnstic uniformity throughout all the lower Vertebral. ^

Why the acid-secrctinp cells of the Vertebrate stomach .u,
free from chlorides during rest and charged with them <lui.
ing activity is a question to which no answer can as y.i l„
given. Doubtless the ijermeability of the cells to chlorides din-
ing activity is much greater than during rest, but how in,
change in permeability is effected is unknown. Kqually diili-
cult to explain are the facts that the acid set free in the cdls
normally moves in one direction, and that every trace of »},.'
acid is swept out of tho cells, immediately it is formed. It is evi-
dent that physical forces, of the nature of which we at present
ha\e no conception, play a \ery important part in these nnt-
cesscs.

V. Summary of Result.s and Conclusions Therefrom

The results of the application of the microchemical molh-
ods of invest'gaiion described in the foregoing pages ma\ be
summarized as follows:

1. Phosphates, and very probably carbonates, are \t>iy
abundant in the cytoplasm of the parietal cells of the Riistric
tubules of the fundus region of the resting Mammalian .stom-
ach while chlorides are absent from these cells, or occur in
them in traces only.

2. Chlorides, phosphates and, very probably, carbonates.
are very abundant in the cytoplasm of the parietal cells of
the active Mammalian stomach.

3. The chief cells of the glands of the fundus of the Mam-
malian stomach are practically free from these salts at all
times, that is, during rest and activity.

4. The phosphates and carbonates and also the chloridos,
when the latter are present in the parietal cells, are cither
distributed uniformly throughout their cytoplasm or, less
commonly, condensed about their nuclei.

Cou.ii': Formation of Hydrochloric Acid

39

.'). The int'istitial tissue of the mucosa of the fundus of
•hv Mammalian stomach is rich in thest- salts at all times and
condensation of these salts occurs in the lymph on the im-
mediate surface of each peptic tubule. I'pon this supply the
ji.iiftal ceils may draw as their activitifs determine.

<i. The u.se of the dye Polychrome B demonstiates that the
)!. -plasm of the parietal cells in certain st^iges of activity is
.111 in reaction, probably owing to the presence of sodium di-
-ydro^en pho.ephate.

7. Phosphates and. very probably also, carbonates are pres-
nt ill great concentrations in the cells of the ga.stric tubules of
hr resting proventriculus of the fowl and of the fundus region
;i the stomach of the turtle and frog. They are confined to
and uniformly distributed in the half of each cell adjacent to
ihi' lumen, while chlorides which are present to a slight extent
■V in traces only are uniformly diffused throughout the cyto-
pl;i>in of these cells.

S. Chlorides, phosphates and very probably also, carbon-
att are present in great abundance in the cells of the glands
lil the active proventriculus of the fowl and of the glands of
the fundus . )rtion of the active stomach of the turtle and
Irop:, but their distribution in each cell is confined to the cyto-
plasm adjacent to the lumen.

9. The results of the injection, into the circulation of the
fiiwl, of a "balanced" mixture of sodium ferrocyanide and
ammonium ferric citrate indicate that hydrochloric acid is
liberated as such on the luminal border of the cells in the gas-
tric priands of the proventriculus of the fowl.

1(1. The foregoing facts give very strong support to Maly's
theory of the formation of hydrochloric acid in the gastric
mutosa. This theory postulates the interaction in the active
acid-secreting cells of the sodium chloride and sodium dihy-
drogen phosphate, the products being hydrochkiric acid and
Jisodium hydrogen' phosphate which latter is converted, as
soon as it is formed, into the acid phosphate by the action of
carbonic acid. The mono-sodium carbonate or the disodium
carbonate which results must quickly pass from the cells into

(J

•10

CoLur: Formation ok Hydrochloric Acid

tho lymph and blcod as its prt'si-nce in the cells wtuiM v
onto terminate the reaction. The cells thus direct tlu. ,, H
they form in one direction, and the alkaline salts lesultniL-
fn.ni the reaction in anotlwr. How this is done is unknown
11 surface tension forces are not involved. Equally unexniaii,'
able are the absence of chlorides dunng rest, their conanti ,
tion during activity in these cells, and the remarkable end, li
satioii at all times, in them, of j-hosphates. Physical l'.„,v.
are doubtless, involved which have not hitherto been f.m.ui
stnking'ly exemplified in n.n-living matter, perhaps beca.is.
the conditions necessary to their manifestation do not .«, u,
therein.

I wish in conclusion to express my thanks to Proftssi,.
Macallum for the facilities provided in the Biochemical Uhuv-
atory of the Univvi^sity of Toronto for this research and .N )
for his kind supervision and helpful suggestions thioujrhmii
the course of the same.

CoLLiP: Formation or Hydrochloric Acid ii

KXIM.ANATION OK PI.ATF.S

I'llOTCX.RM'lls

I. Transverse s«'<ti(»n of the imu nsa <<( the IiiikUis ,if tin u ti\tl\
.in ting stomach of doR. Chlnritlcs, plin-iihalr-. am! < arUjiMtts

\
tnwii III till- parutal nils. -j — ArNO, -low tnaKiiilii atioii.

J. A portion of same nion- liifthly nia(;nifii-<I.

:!. TransvtTst' sf( tion of tlir gastric tiiinilis in tlic fmidii^ of the
•i.iitivo stomach of rat. Chlorides shown coiKJiiisid in the

N
lUTstitial tissue. j-. ArNOi+I..') per (cnl. II.VO,.

I. Transverse section of the tulniles in the active provenirit -
iliis of hen. Chlorides, phosphates and carlH)nates loc.ili/e<l.

„. ArNO,.

Drawint.s

1. Transverse sct'tion of a jjastric tubule in the fundus region of
!'i( active stomach of dog. Chlorides loca!i/e<l in the parietal cells.

\

III .\kNO, + 1..'5 per cent. UNO,. x 1 120

2. Transverse section of a tiilmle of one of the multilobular

i;ldii(ls of the active proventricixlus of hen. Chlorides, |)hosphatcs

N
ind carbonates localized. AgNO^. xll20

:{. Transver.se section of a tubule of one of the multilobular

glands of the active proventriculus of hen. Chlorides alone

N

I'liahzed. — AgNOj+l.S percent. HNOa. x 1120

1. Transverse section of a tubule of the inactive proventriculus of

42 Coixip: Formation or Hywochloiiic Acid

l\«n Clilori.lix, pho-iphatiH ,111(1 ,.irlMin.it«s|iM.ili/t(| \ \, ,

III

Ut\t

X 1 :.'ii

"i. Tninsvcrst' M«itiitn <.f a nastri< iil.iilf ..f thr inarti\. |.,,,

vrntrinilus (if Ikri. (hloridis alom- ari- slinwn, -— AjjNO, ( i",
fXT r«-nt. UNO, ^ ,1 .„

Hern

«. TransviTsf Mctinii of a gastric tiilmlc of tin- funtltii rcni. i, , •
till- inarlivf ^toiiiai h of tunlf. Clilorulis. phospdatt s an.l carl .r

Beau

atw I.Kali/t.,1. ,j, AkNO ,,,.,,

Rrov

7. Traii^viTM' s«-<lion of a trastric tulnili- of th^ fun. In, r..,/,,...

Brur

the

iiuutivi- stoniath of fr.ig. Chloridi-s, pliosph,

■» ri'sfiiiii f

itfs .111(1 cirl

atex liK-alizi'd. -- AgNO;

10

% ll.'o

S. TransvtTSf .st( tion of the terminal jjortion of a tiil»ul

active proventriculus of a hen whi( h had

e..f !|i,

received an iritr iv

injertion of a "balanced" mixture of sodium ferrocyanid.
ammonium ferric citrate. The pr.HJuction of hydrochl
demonstrated by the <ieposit of Prussian blue.

oric .11 hi
X ll.'O

COLLIP: Formation ok nvDiKKiM.oRic acid

i:i

nilU.IOCUA 'HV

Btn«ley, H. R 18M— The Struiture of thi- Muiiuiittllan (luxtrir

Glarnl. Quurt. Jour, of Miiro. Sc . Vol.

41. p. Hfli :;8;),

1SH)0 Thi- tk.sophuKPuI Clitml. of Uro<l»la.
Biol. Hullttin, Vol. 2, N... !. p 87 lo:t.
lie riiurd, Claude 185»— l^r^ons >-ur le. I'ropri^ti'. !'hy,«iolom«|u»ii

(lox Litiuidf- (Ir I'Oiiftiiii-tin'. Purio. p.

a7ii.

Bfuumont , ISn.T- Ol)«erviitions of thr (ia-liu Jui.f. I'lalt^-

burKh.
Rrown, C. P 1912— On th» di.'lribution of potu--ium ip renal

<ell>. Truno. Cm. IriKt.. p. :i8!t.
Brurk* 1851»— Sitz. K. Aku<l. d. Wiv.. \U\. V.T.

H-iJ'i-'*'. a 1889— LrhrLuoh der Chftiiic, Lfipzii;.

'"jii". M 1888— Kerherchr-' An»tonii(|ue.«, Hi-tolo|{i(iue^

et KnibryoloKiqucK sur rAppnri'il (la.i-
trii|ue (Ics Oi-t'aux. Annal. <!«•■; Sricnrc-
Nutur. Zool., 7 Scrip, Vol. 4, p. m-:m.

Kb-tein ond Griltiner 1872-Ufb<'r den Ort der I'cpMn-nilduni? ini

MuKi-n. PflUKc?r'.- Arch., ltd. f., S. 1-18.
1874 -Lcbti Pt-psin-BdilunK Ini MaKen, Pflujfei'-
Arch., Bd. 8, S. 122-151.

K.lipiTPr, L 1879— Zur Kenntnis dcr UrU^onzclIcn dps Maif-

■en.s besondcrs beini Mon.-rhpn. Arch. fUr
Mikro.-ik. Anat , Bd. 7. S. l'J3-211.
1882 — Uebcr die Kcaktion der Ubcndon Maitcn-
schlcimhuut. PfliiKcr'.s Arch. Bd. 29, S.
247-256.

litiGerald, M 1910— The Orih-in of the Hydrochloric Acid in

the Gastric Tubule.s. Proc. of the Roy.
See, Vol. 8:3, B. p. 56-93.

Friinkel, S 1891 -BcitraKC zur Phy.-ioloKie der Ma)?en-

drUsen. PHugcrV Arch., Bd. 48, S. 63-73.

Gilbert, S 1898 — Die BcstimmunK de.s Kaliums nach Quan-

titativer Ab>chcidunK de.->elbep al.s Nat-
rium Cobalti Nitrit. Inaugural Disserta-
tion, Tiibinjfcn.

''""^'''i. S 1902— Untersuchungen Uber die Ma^enver-

dauung neuReborener Huiide. Pfliigcr's
Arch., Bd. 90, S. 591-615.

u

11

t'oLLif: Formation or IIvmukiiloric Acid

UrvMiwowl, M 1885 - Ob>rrvntion un th» <U«trlo JJInn.!- ..r t.^

I'iK. Juurn. of i'hy>ioloiry. Vol. fi, « | .

288.
Hamm«», F. S. 1915 -Th«> Sour.e ..r thr Hydrothlorif \, i

found in the Htuiiiitch. Anal. lU.ut.i

Vol, 'J. No. I, |i. 21 2.'..
Harvry, B. C. II., ftnd ~ U|H>n thr formiitioii of th* H>.lroa,i,

Ucnulcy, n. U. 1912 AtitI in thr FovihiIhi- un<l on the Surt , .

i)f the (i(«»trl<- Murou>i Mi'iiibrane, aiut t^.

Nan- Aril) ("hnrui'trr of iho t'oiittril .

the (flan«i C'flU nnil l.uminn. Hiol. iluj! ,

Vol. 2:), No. 4. p. 225-240.
HBKie, C IBfifl IWitrttKt «ur Hi.stologic <!<■» Vonelmaif.n-

Zfit . f. Nut. Mel., H(|. 28. Heft 1. p I
HeiUfnhaln, R 1870— Untersuchunjfrn uber .len fiuu rlir I ,'

(IrU.fn, Airh. fUr Miliro-.k, Ani«t,, H.| i.

S. WH.

Klug, S 1893— Pie H.ltKullfn .kr Matfpn.^.hlei.nli.iut

biMen au»»«>r ikr Stt'Tc Hurh da.'i Pep. n
Unjcar. Arc-h. f. Med.. > 15.

Lungley, J. N 1881— On the Hi^tolojfy or.' ! hyuiolofry -f th.

Pepsin Foriiiintt Glundx. Phil. Tian-. of
the Hoy. Soi-., Vol. 172, p. 06.1-711.

Langley, J. N., Mid

Scwall, U

Lepine, R

Macallum, A. B.

1879_-Journ. Phyj<iol., Vol. 2. p. 284.
.1872— Kxperiencesi mr le» Glande.H de I'L'.to-

niBo. Compter Rendus et Mem. de l.i $•„■

de Biol., Vol. 24, p. 221.
..1898— On the Dcteotion and Localization vf

Pho!tphoru.H in Animal and Vetfetahlf Ti

sues. Proc. of Roy. Soc., Vol. 63, p if,:-

479.
1905— On the Distribution of PotajiMum in An

mal and Veifetable Cells. Jour, of

Phy^iol.. Vol. 32 p. 95-123.
1905— On the Nature of the Silver Rea< tion in

Animal and Vegetable Tissue.^. Proi. of

Roy. Sor., Vol. 76B, p. 217-229.
1908 — Die Methoden und ErKebniKiie der Mikro-

chemie in der biolot^isohen For.-iohunif

Ergebnisse der Phy.siolo^ie, Bd. 9, S. 1.32-

6M.

CwxiP: Formation or HYtWfK iii.oRic Acid

»••

V ilv, R » .. 1874- Untfr.urhunK»n uber ili* gu»IIv <l#r

Mavcn-iiartaKurc. l.tcbigV AnnaUn. Bil,

»7n. H. 2fio.ar.7.

1171 — Untpr»urhuni{en ub«r die Mitti-I «ur

HttUreblMunK im Or«rtni.ritu- Zeil-.h

fUr rhy>i«l. Chemie, B.I., I, S. 325-3.16.
Moiin __.._ l*JM>— Suuh »timi«rhi <IvkIj ucoellj. Itenkurhrtf-

l»n rler Wurirr Akitii. math nnturwiKi.

Kta».e III , II.I, 2, Al.t. 4. p. 24.
N'oiirden, C. von l«8fl -MuKfn.<Mrt.'-<>fr«tion und Hlutulkal«>o«ni.

Arih fUr Kx|m>i-. I'nthol, urul l'h»rniak«»l,

n.1. 21 and 22, S. .1?.V,1!1«.
N'u<«b»um, M 1878— UrlxT drn lluu und die Thtttiifkeit diT

DrU»«n. Anh. fur Mikro^k. Anal., Bd.
1 15, S. 119.133.

1879-Ueb«r dtn Bau und dip Thtttltfkcit d»r

l>rU..«)n. Arch, fur Mikro.-k. Anat,. Hd.

Ifl, S. .V12.
OpP«l. A 180«5— Lthrbu.h d VcrRl. Mikro,kopUrh«n

Anutomic, Teil I. Jfna.
Partsch, K. 1877— Ueitruifp lur Kcnifni* di?.>« Vorderdarmfs

einiiter Amphibi«n und Rcptilien. Arch.

f. Mikro.sk. Anat., Ud. 1, S. 179-203.
•'■""«. W 1824— On the Noture of the Acid and Snlin*

Matter u.sually Kxi«tinu In the Stomach

of Animalii. Phil. Trann. of Roy. Soc,

Vol. 144, p. 45-49.

Riaumar 1752— M.-m, de I'Acud. des. Soi., p. 226.

R«>«h. J 1874— The Acidity of the Gastric Juice. Journ.

of Anat. and Phys., Vol. 8, p. 274-284.
III. hot, C 1878— Des Proprii'tts Chimiquen ct Physiolo^-

ques du Sue Gastrique chc« THomme et

le.'< Animaux. Jour, de I'Anat. et de la

Phyjiiol.. p. 170-326.
Ko-cmann, B 1907— Beitrftue »ur Phyaioloifie der Verdauunif.

PfluKer'.s Arch., Bd. 118, S. 467-624.
Sehrwald, E 1889 -Die Beletrzellen des Mascens al.', Bildun(^

statten der Saure. MUnch. Med. Wochen-

Kchr., No. 11, p. 177-180.
Swiecieki, H 1876— Untersuchunjren uber die Bildunn und

Au.sschcidunK des Pepsin.s bei Batra-

chien. PflUifer's Arch., Bd. 13, S. 444-452.

Vr»

4«

COLLir: rcilMATION or ilYDRUCHLOHIC ACU)

iMbe, I>

Utintiint, „...„

TriBkl»r. N .„.
Witt. O „

U77

IH'J

ISItt

ll»ii

ll«ilr«icii tur Kcnntni. lUr trm^^n «««,,
d»« M*n.ihltx-h*n Ma(»n**ft«>, JU,i,. >,
I. I'hjfiiol. Ch»ml». Bd.. I, g. Uinrrfl
Zum >'«ln*rtn Rmu unci Mr I'hy^oi.,,^ .
•l»r M>ff«nr.'hl*inihiiui MUn<h M* i
W<xh«n»rh , H. 7t>3.

Uelirr .Iffi ilau .J^r Magwi.rhUio I,,, ^t
Areh t. Mikro.k. An«t.. iM. U, S ITj
310

l'»b»( iliF Cynniinii*. cinr h«ue Ciuppr
von Kurb.loffrn Her il d. rh»tti (W \„
2.1, p 22-47

I'l'"li nil. l.i;;l,i|ili \,. I

rili'l.i iiii.n',;i.i|.|i \,,. J

;!

^

MiiMI

rilriln-lliii ioi;r,i|,|\ .\

niMpii .No. .;

riiot()-niicrogr,i|ili NO. (

a

Prawing No. 1

Drawing No. 2

KfJj

i

l>rawing No. 3

Drawing No. 4

. 'Ift ' *

,j ^ ••-« -ijr ..|r"iS«iiii

IVawiiiK No. 5

Pr.iwinc \<i. rt

0]

vV

Drawing No, 7

I'r.iviing Nil. S

j^^^^km

So
No

v..

No
N...
No.

No.
No.

No.

So.
No.
No.

No.

No.
No.
No.
No.

No.
No.

No.

fNIXI'KSIIN- 0\ IDKONIti M 11)11 ^

riu>i.'i HI. I, \i Ni Kii ,

So. i: 111,* olriuliiti'. ini^r.i-. lii-iiii-iM .in.l tli \ ilr|ii>Kiir c!
licrvi-cells, Willi >(>.xi.il iit.iiiii, I,, iluir nil, U, II ,,,„i.
poiiiiiN, h\ I- II Si ,11 I ,i .^

No .• : i>ii the . >l»l,>t,'\ ,i| ii.>M.iMnU-.ili,l .■i-.iili-inx h, \ I;
M \l \l [ i M

" 7 "
\o. (: Oh^,■l^.ttl>■llN ,Mi hlo.Hl pi,>,iii,-, h, H |i Kii,,,,! ,,. -•

\» i rill' ,hfnll^tr> ,.l whc.it >;l,ili'ii, In i, t, \\,miiii u.\i<

No 5: The p.il.ii',i,luitiislr> i>l llii >•>,•. 111. in \ 1; \| u Mil \, ,, _. .

No. ii: riu- abs.>rpii,iii ,.| |.,i „, n,,.. ii,i,.,i 1,,,.^ |,^ i, | \\ ,^„^ __ .^^

Nil. 7 : riic ilisinbiilKiii ,i| l.u. , I1I..1 ulv., pli.'^pti.ii,^. p,.t.,-,i;iMi

anJ irmi in «.lri.ileJ iiiiis,!,', h\ M \i 11 1 \Iimis ,,^-

\,i. H : SurLuetciisiDii .iiij vii.il pliiii.Miicii.i. h\ \ II \| \, u 1 1 m 1 , k,
N,i. (, : On tho iliNlrilniliKii vl p,i|.issiiiiii m un.il .vU* In

C. p. HK,m\ .',...-

.Ni>. 10: On the pr,i|i.il>le iiatiiu' .it tlu' MihsinK,- pi,.iiu.im^

t,'rowth m Viiiinj; .iDiiii.tU, In I \-imiu I 1 \k .,,,J \ |Ihi,7

Mai ai.I I'M

N.'. ri: The i-ump.initivc valii.' ,i| l,ii,l aiul hutt^'i m -i.nuli,

by Casimik Im \k .nul .\. Uki , i- \I \. m n m " ■ .. -• i

No, 12. The a, lion ,'l yci^t fra,tiaiiN ,iii iIk ^;i.u\tli .'I i.iis. In

Casimih Kt.vk aii,l .\. Mhi.i Mmviiim ^,_.-

No. I'?: A new on, epluni ,i| ili,- >;I,-iir'i ul.n liiii,ii,Mi. In,
T. (i. l!l<iM'll'

Ou clianf,'i-s 111 thf j;l,iiiicriili aiul luhul,., ,,| ilu- ' ""
kidney accompanyiii^' a, livity, h\ I", d. I!k,'|i|i iukI | | I
Mai kis/ik I

No. 14: Further ob.sorvalioiis ,.ii iIk- tlilK r,.iiti.il a,tioii ,.|

adrenalin, h> I-'kank A. IIahimw ,111, 1 Ivi- .M, I'm nu vs I
No. 15: The meclinnism tor \asodilatalion lr,>m a,lrin,iliii, In 1 "■ .'i"

FkA.sk a. HAKLMANaiul I.Ols Ml I'll! I l|j.\\ ||;\s|k '

Niv |(): Adrenalin vas,KliIator flieihaMisiiis m tli,- ,.it at iliHer, iit

a>,'es. h\ Fka.vk .\. II.vkimas and l.i.-i 11 I.. Kiiiun\ ., j^

Ni>. 17: Location ot tlie adrenalin \as,Hiil.il,M iiulIi.iiuniiw, In

Fhask a. lIxKi.MA.s, I.. G. Kii ii.ti(\ and l.,>i Fx \-i u ..j^
No. iH: Vascular i.lianj,'es prodimd hy adrenalin in M-itehi .it,s,

by Fkank A. Hakima.v, Lk-i 11: li. Kii imks .md K,.s^ >

I. am;

N,.. ii)t Siniplilied f,'a-. analysis, hy J. |. U. M\, ii,i|i

No. Jo: .Adrenalin vas,idilator nie, hanlsins. b\ Ik.wk .\

Hahtman, Lfsi.IH li. Kii ii,.K\ and I..ms I'kami;
^o. 21 : Constriction from adrenalin actiiii; upon s\ mpatli.,tic

and dorsal root f,',nitjlia, by Fkank .A. MaimmW. I.i s| .,

Ci. Kll.HOK.v and Lois I"i<as|-k

"■ -'.-1

N»>. Ji I iti -['""It.fv'.'ll-. vl. \ -ii'l lll-.'M' ."t i:| 1, ).|>>«»» .mullltOIr

lit .Uufvbr.il. . lU, i»- I i K Mi.ifiMt o.ls

\,. ; i In.' Ji.uiu>-i- .1 KiJ".-. kr> I I U \|v. i..«i» i>l*>

\.. .'I Suiiplitutl c '- " I I U M V. 1 ■ >"> "J"

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