agent, pepsin can act only in the presence of acid, and we have every
reason for believing that the enzyme and the acid form a compound,
which in turn combines with the proteid undergoing digestion; or, what
amounts to much the same thing, that the acid perhaps forms first a
compound with the proteid, to which the pepsin can then unite to form
a still more complex compound capable of undergoing hydration and
cleavage. Pepsin-proteolysis, therefore, is strictly the proteolysis
produced by pepsin-acid. In view of this fact, we may well give a
moment’s thought to the nature and origin of this acid.
Without attempting any statement of the gradual development of our
knowledge regarding the acid of the gastric juice, we may accept the
now well-established fact that the acid is hydrochloric acid, and
that it has its origin in the parietal, or so-called border-cells of
the gastric glands. That the acid is derived from the decomposition
of chlorides is practically self-evident, but Cahn[94] has added
experimental proof which removes all shadow of doubt, through his study
of the gastric secretion in animals deprived for many days of salt; the
gastric juice in such cases being perfectly neutral in reaction, but
normal as regards its content of pepsin.
[94] Die Magenverdauung im chlorhunger. Zeitschr. f. physiol. Chem.,
Band 10, p. 522.
The way in which the specific gland-cells manufacture free hydrochloric
acid out of material contained in an alkaline medium is somewhat
doubtful. There are, however, at the present day two theories worthy of
special notice. The first is based upon observations made by Maly[95]
many years ago, which tend to show that certain mineral salts present
in the blood are capable of reacting upon each other with formation
of hydrochloric acid. Thus, while the blood is an alkaline fluid,
it really owes its alkalinity to the presence of two acid salts,
viz., sodium bicarbonate (HNaCO_{3}) and disodium hydrogen phosphate
(HNa_{2}PO_{4}). This latter compound, acted upon by the carbonic
acid of the blood, is transformed into a dihydrogen sodium phosphate
with simultaneous formation of acid sodium carbonate, as shown in the
following equation:
Na_{2}HPO_{4} + CO_{2} + H_{2}O = NaH_{2}PO_{4} + HNaCO_{3}.
[95] Untersuchungen über die Quelle der Magensaftsäure. Annalen d.
Chem. u. Pharm., Band 173, p. 227.
This acid sodium phosphate dissolved in a fluid containing sodium
chloride, gives rise to free hydrochloric acid by a very simple
reaction:
NaH_{2}PO_{4} + NaCl = Na_{2}HPO_{4} + HCl.
It is also to be noted that the disodium hydrogen phosphate, may,
likewise, give rise to hydrochloric acid through its action on calcium
chloride, as indicated by the following equation:
2Na_{2}HPO_{4} + 3CaCl_{2} = Ca_{3}(PO_{4})_{2} + 4NaCl + 2HCl.
It is thus evident that hydrochloric acid may originate in the
inter-reaction of these several salts which are known to be present
in the blood; but obviously, the above reactions cannot take place
in the blood itself, and we must look to the selective power of the
epithelial cells of the gastric glands, as suggested by Gamgee,[96] for
the withdrawal of the needed salts from the blood. Once present in the
acid-forming cells, and perhaps aided by the inherent qualities of the
protoplasm, the necessary chemical reactions may be assumed to take
place, after which the newly formed acid may pass from the gland-cells
into the secretion of the gland.
[96] Physiological Chemistry of the Animal Body, vol. 2, p. 113.
A later theory regarding the formation of the acid of the gastric juice
emanates from Liebermann.[97] This investigator claims the existence in
the mucous membrane of the stomach of an acid-reacting, nuclein-like
body, which is apparently a combination of the phosphorized substance
lecithin with a proteid. To this compound body Liebermann gives
the name of lecithalbumin. It is apparently located in the nuclei
of the gastric cells, is strongly acid in reaction, and, according
to Liebermann, is an important agent in the production of the free
hydrochloric acid of the gastric juice, although its action is somewhat
indirect. According to this theory, the free acid is formed in the
mucous membrane of the stomach from sodium chloride, through the
dissociating action of the carbonic acid coming from normal oxidation.
The thus-formed acid then diffuses in both directions, viz., through
the lumen of the gland into the stomach-cavity, and in part in the
opposite direction into the veins and lymphatics. It is the assumed
function of the lecithalbumin to react with the alkaline sodium
carbonate, produced simultaneously with the hydrochloric acid. This
naturally gives rise to the liberation of carbonic acid and to the
formation of a non-diffusible sodium-lecithalbumin compound, which
is retained for the time being in the body of the cell. When the
circulation of the blood, accelerated by the digestive process, returns
to its ordinary pace, this latter compound is slowly decomposed by
the carbonic acid with formation of the readily diffusible sodium
carbonate, which passes into the blood-current. The rate of this
latter reaction is impeded, or, perhaps regulated, by the swelling up
of the lecithalbumin-containing cells, thus rendering the imbibition
of the carbonic acid a slow process. The rate of production of the
hydrochloric acid by this hypothetical process depends primarily upon
the blood supply, and the oxidative changes by which carbonic acid is
formed.
[97] Studien über chemische Processe in der Magenschleimhaut. Pflüger’s
Archiv f. Physiol., Band 50, p. 25. Neue Untersuchungen über das
Lecithalbumin. Liebermann, Ibid., Band 54, p. 573.
There is much that might be said for and against this theory,[98] but
we cannot stop to discuss it here. Like the previous theory, it implies
the production of hydrochloric acid from a chloride or chlorides,
through chemical processes taking place in the stomach-mucosa, and
presumably in the large border-cells of the peptic glands. This
hydrochloric acid, as you know, in the act of secretion, reacts upon
the pepsinogen with which it may come in contact, transforming it into
pepsin. It also has the power of combining with all forms of proteid
matter, not excepting the products of proteolytic action, to form acid
compounds in which the so-combined acid, although equal quantitatively
to the original amount of free acid, is less active in many ways.
Thus, it does not possess in the same degree a destructive action on
the amylolytic ferments;[99] it does not play the same part in aiding
the proteolytic action of pepsin, and its antiseptic power is far from
equal to that of a like amount of free acid.[100]
[98] See discussion by Plósz and Liebermann in Jahresbericht für
Thierchemie, Band 22, p. 260.
[99] Chittenden and H. E. Smith: Studies in Physiol. Chem., Yale
Univer., vol. i., p. 18.
[100] Compare F. O. Cohn: Ueber die Einwirkung des künstlichen
Magensaftes auf Essigsäure-und Milchsäuregährung. Zeitschr. f. physiol.
Chem., Band 14, p. 74.
With relatively large amounts of proteid, we may have half or even
quarter saturated proteid molecules, in which the weakness of the
combined acid is far more pronounced than in the case of the fully
saturated molecule. Such a condition of things must obviously exist
in the early stages of gastric digestion. With an excess of proteid
matter in the stomach, some time must elapse before the secretion
of hydrochloric acid will be sufficient to furnish acid for all of
the proteid matter present, yet pepsin-proteolysis does not wait the
appearance of free acid. Indeed, the proteid matter may not have
combined with more than half its complement of hydrochloric acid before
digestive proteolysis is well under way. I have made many analyses of
the stomach-contents after test meals, and under other conditions,
where no free acid could be detected by the tropaeolin test, or better,
by Günzburg’s reagent (phloroglucin-vanillin), although phenolphthalein
as well as litmus showed strong acid reaction, and yet not only could
acid-albumin be detected in the filtered fluid, but likewise proteoses
and peptones. In other words, pepsin-proteolysis can proceed in the
absence of free hydrochloric acid, although not at the same pace.
Hence, proteoses and even peptones may make their appearance in the
stomach-contents at a very early period of digestion, _i. e._, the
final products of proteolysis may be found in a mixture containing even
a large proportion of wholly unaltered proteid, and obviously at an
early stage in the process. Expressed in other language, a portion of
the first formed acid-albumin or syntonin may be carried forward by the
digestive process to the secondary proteose and peptone stage, before
the larger portion of the ingested proteid food has even combined with
sufficient acid to insure the complete formation of acid-albumin.
This introduces another factor, to be referred to later on, viz.,
the relative combining power of different forms of proteid matter,
especially the proteoses and peptones, as contrasted with native
proteids.
In proof of the statement that pepsin-proteolysis can proceed in the
absence of free hydrochloric acid, provided combined acid be present,
allow me to cite one or two experiments bearing on this point. A
perfectly neutral solution of egg-albumen, containing 0.8169 gramme
of ash-free albumin per 10 c.c. of fluid, was employed as the proteid
material. In order to completely saturate the proteid contained in
20 c.c. of this neutral albumen solution, 50 c.c. of 0.2 per cent. HCl
were required. Two mixtures were then prepared as follows:
_A._ Twenty c.c. of the neutral albumen solution + 50 c.c. 0.2 per
cent. HCl + 30 c.c. of a weak aqueous solution of pepsin, perfectly
neutral to litmus. This mixture gave only the faintest tinge of a
reaction for free acid when tested by Günzburg’s reagent.
_B._ Twenty c.c. of the neutral albumen solution + 25 c.c. 0.2 per
cent. HCl + 30 c.c. of the neutral pepsin solution. In this mixture,
the proteid matter was obviously only half saturated with acid.
The two solutions were placed in a bath at 40° C., where they were
allowed to remain for forty-four hours, a little thymol being added to
guard against any possible putrefactive changes. At the end of this
time the amount of undigested albumin was accurately determined. The
20 c.c. of original albumen solution contained 1.6338 grammes of dry
coagulable albumin. At the end of the forty-four hours, _A_ contained
only 0.5430 gramme of unaltered albumin, or acid-albumin, while _B_
contained 1.2225 grammes. That is to say, in the mixture _A_, where the
acid existed wholly in the form of combined acid, but with the albumin
completely saturated, 1.0908 grammes of the proteid were converted
into soluble albumoses and peptones. In _B_, on the other hand, where
the albumin was only half saturated with acid, 0.4113 gramme of the
proteid was converted into soluble products. This difference in action
is made more striking by the statement that where the proteid was only
half saturated with acid, 25.1 per cent. of the albumin was digested;
while with a complete saturation of the proteid, 66.7 per cent. of the
albumin was digested.
To give emphasis to this matter, a second experiment may be quoted as
follows: The proteid used was the same neutral solution of egg-albumen
containing 0.8169 gramme of albumin per 10 c.c. Two mixtures were
prepared as follows:
_A._ Ten c.c. of the neutral albumen solution + 21.7 c.c. 0.2 per cent.
HCl, the amount needed to completely saturate the proteid, + 40 c.c. of
a weak solution of pepsin, perfectly neutral.
_B._ Ten c.c. of the albumen solution + 10.9 c.c. 0.2 per cent. HCl +
40 c.c. of the pepsin solution, making a mixture half saturated with
acid.
These two solutions were warmed at 40° C. for seventeen hours. The
extent of digestive action was then determined, when it was found that
in _A_ only 0.1638 gramme of the proteid was undigested, while in _B_,
0.6088 gramme remained unaltered. In other words, where the proteid was
completely saturated with acid, but with an utter lack of free acid,
79.9 per cent. of the albumin was converted into albumoses and peptone,
while in the mixture half saturated with acid only 25.4 per cent. was
digested.
These two experiments thus give striking proof that free acid is not
absolutely essential for pepsin-proteolysis. Digestion is, to be sure,
more rapid and complete when free hydrochloric acid is present, but
proteolysis is still possible, and even vigorous, when there is a
marked deficiency of free acid. Further, as we have seen, proteolysis
may proceed to a certain extent even though the amount of acid
available is not sufficient to combine with more than half the proteid
matter present.
These facts at once raise the question whether the products of
proteolysis may not have a stronger affinity for acid than the native
proteids; an affinity so strong that they may be able to withdraw acid
from the acid-albumin first formed. One of our conceptions regarding
pepsin-proteolysis is that acid is necessary for every step in the
proteolytic process. A primary albumose, for example, cannot be further
changed by pepsin, unless there is acid present for it to combine
with. This being true, it is clear, in view of the fact that even
peptones may appear in a digestive mixture containing an amount of
acid insufficient to combine even with the albumin present, that the
products of proteolysis must withdraw acid from the acid-albumin first
formed. In regard to the first point, my own experiments certainly
tend to show that the products of gastric digestion do combine with
larger amounts of hydrochloric acid than undigested proteids; and
further, that of the several products of proteolysis, the secondary
proteoses combine with a larger percentage of acid than the primary
proteoses, while true peptones combine with still larger amounts. In
other words, the simpler and more soluble the proteid, the larger the
amount of acid it is capable of combining with; a statement which
accords with results obtained by other workers[101] in this direction.
Further, another factor of considerable importance in connection
with the natural digestive process is that a dissolved proteid, such
as protoalbumose for example, will combine more readily with free
acid than an insoluble proteid; from which Gillespie[102] is led to
infer that in pepsin-proteolysis where there is no free acid present,
only acid-albumin, proteoses may be formed to a limited extent at
the expense of some of the acid of the acid-albumin, a portion of
the latter being perhaps reconverted into albumin. The ability of
the proteoses, however, to withdraw acid from its combination with a
native proteid is perhaps best indicated by Kossler’s[103] experiments,
which show that a solution of acid-albumin containing only enough
hydrochloric acid to hold the albumin dissolved, on being warmed at
40° C. for some hours with addition of a neutral solution of pepsin,
may undergo partial conversion into albumose or peptone.
[101] See especially Gillespie: Gastric Digestion of Proteids. Journal
of Anat. and Physiol., vol. 27, p. 207.
[102] Loc. cit.
[103] Beiträge zur Methodik der quantitativen Salzsäurebestimmung im
Mageninhalt. Zeitschr. f. physiol. Chem., Band 17, p. 93.
In spite of these facts, there is some evidence that while proteoses
and peptones have the power of combining with more acid than a like
weight of native proteid, the latter, leaving out all action of the
pepsin, has a stronger affinity for the acid; in fact, the firmness
or strength of the union appears to diminish as the products become
simpler.[104] Hence, a peptone separated from a digestive mixture, will
part with its combined acid somewhat more readily than acid-albumin
for example, although on this point there is not complete unanimity
of opinion.[105] In digestive proteolysis, however, where the pepsin
is accompanied by a minimal amount of hydrochloric acid, insufficient
perhaps to even half saturate the proteid present, the formation of
proteoses and peptones must be accompanied by a withdrawal of acid from
its combination with the native proteid.
[104] Compare Blum: Ueber die Salzsäurebindung bei künstlicher
Verdauung. Zeitschr. f. klin. Medicin, Band 21, p. 558.
[105] See Sansoni: Beitrag zur kenntniss des Verhaltens der Salzsäure
zu den Eiweisskörpern in Bezug auf die Chemische Untersuchung des
Magensaftes. Berliner klin. Wochenschrift, 1893, Nos. 42 and 43.
In illustration of some of these points, and especially of the
statement that the products of gastric digestion have the power of
combining with more hydrochloric acid than the original proteid, allow
me to cite the following experiment: 10 c.c. of a neutral solution of
egg-albumen containing about 0.82 gramme of pure dry albumin, free
from mineral salts, required 23.8 c.c. of 0.2 per cent. hydrochloric
acid to completely saturate the proteid matter. A mixture was then
prepared as follows: 10 c.c. of the albumen + 24 c.c. 0.2 per cent. HCl
+ 30 c.c. of a neutral pepsin solution, the mixture showing a faint
trace of free acid when tested by Günzburg’s reagent. This solution
was placed in a thermostat at 38° C., and from time to time a drop of
the fluid was removed and tested for free acid. If no reaction could
be obtained, 0.2 per cent. hydrochloric acid was added to the mixture,
until Günzburg’s reagent showed free acid to be again present. The
following table shows the rate of disappearance of free acid, and the
amounts of 0.2 per cent. HCl required to make good the deficiency.
The mixture was placed at 38° C. on February 6th, at 11.30 A.M., and,
as stated, contained a trace of free acid, 24 c.c. 0.2 per cent. HCl
having been added to accomplish this result.
Time. Acid added to show trace of free acid.
February 6, 11.30 A.M.
" 2.15 P.M. 4.5 c.c., 0.2 per cent. HCl.
" 5.00 P.M. 1.0 " " " "
February 7, 8.45 A.M. 3.0 " " " "
" 2.00 P.M. 1.0 " " " "
" 5.00 P.M. 1.5 " " " "
February 8, 8.30 A.M. 1.0 " " " "
" 2.30 P.M. 0.0 " " " "
February 9, 8.30 A.M. 3.0 " " " "
February 10, 9.30 A.M. 2.0 " " " "
----
17.0
From these results several interesting conclusions may be drawn,
in conformity with the statements already made. Thus, as soon as
proteolysis commences, the products formed begin to show their greater
affinity for acid by withdrawing acid from its combination with the
native proteid, a supposition which is necessary to account for even
the starting of the proteolytic process. Further, it is evident that
proteoses and peptones combine with a far larger equivalent of acid
than the native albumin is capable of; 17 c.c. of 0.2 per cent. HCl
being required in the above experiment to satisfy the greater combining
power of the newly formed products. This doubtless depends upon the
cleavage of the large proteid molecule into a number of smaller or
simpler molecules, each of the latter, perhaps, combining with a
like number of HCl molecules. This view of the relationship of the
individual proteoses and peptones is one more or less generally held,
and is supported by many facts.[106] However this may be, it is evident
that the products of pepsin-proteolysis combine with a larger amount of
hydrochloric acid than the mother-proteid, and that the transformation
of the latter, at least under the conditions of this experiment,
is a slow and gradual process. In the living stomach, on the other
hand, where the secretion of acid is progressing with ever-increasing
rapidity, it is easy to see that the process of proteolysis would
naturally be much more rapid.
[106] See Gillespie: On the Gastric Digestion of Proteids. Journal of
Anatomy and Physiology, vol. 27, p. 209.
Just here we may recall the theory advanced by Richet[107] quite a
number of years ago that the acid of the gastric juice is a conjugate
acid, composed of leucin and hydrochloric acid, a theory which has
found little acceptance. Klemperer,[108] however, assumed that
solutions of leucin hydrochloride with pepsin would not digest albumin,
but Salkowski and Kumagawa[109] have shown by experiments that leucin
and other amido-acids, as glycocoll, may be dissolved in hydrochloric
acid in such proportion that the solution is practically composed of
leucin hydrochloride, without interfering with the digestive action
of pepsin-acid on blood-fibrin; the solution being physiologically
active, although Günzburg’s reagent shows an entirely negative result
for free acid. If the matter is studied quantitatively, however, it
will be found that the amido-acids combining in this manner with the
hydrochloric acid of the gastric juice do give rise to some disturbance
of proteolytic action;[110] _i. e._, digestion may be less rapid,
especially on egg-albumin, a conclusion which Salkowski[111] has lately
confirmed. Still, under such circumstances, digestion does go on and
at a fairly rapid rate; hence, if there is a combination between the
acid and these organic bodies, as is indicated by Günzburg’s reagent,
the acid is still active physiologically, even more so than in the
compound formed by the interaction of proteid and acid. In other
words, many of these neutral organic bodies that may originate in
the stomach through fermentative processes, or otherwise, and which
tend to combine with the acid of the gastric juice, do not, as a
rule, impede pepsin-proteolysis to the same extent that an excess of
proteid matter may. In fact, in artificial digestions long continued,
pepsin-acid solutions containing considerable leucin, for example, may
accomplish as much in the way of digesting proteid matter as the same
amount of pepsin-acid without leucin; but the inhibitory action of the
amido-acid is there, and may be shown during the first few hours of the
experiment, when less proteoses and peptones are formed than in the
control experiment without leucin.
[107] Le suc gastrique chez l’homme et les animaux, ses propriétés
chimiques et biologiques. Paris, 1878.
[108] Zeitschr. f. klin. Medicin, Band 14, Heft 1 and 2.
[109] Ueber den Begriff der freien und gebundenen Salzsäure im
Magensaft. Virchow’s Archiv, Band 122, p. 235.
[110] Rosenheim: Centralbl. f. klin. Medicin, 1891, No. 39. F. A.
Hofman, ibid., No. 42.
[111] Ueber die Bindung der Salzsäure durch Amidosäuren. Virchow’s
Archiv, Band 127, p. 501.