that the proteinochromogen, which could not be isolated by itself
in sufficient purity for analysis, must contain approximately 61.02
per cent. of carbon, 6.89 per cent. of hydrogen, 13.68 per cent. of
nitrogen, 4.69 per cent. of sulphur, and 13.71 per cent. of oxygen.
As a proteid-like body, it is thus especially characterized by an
exceedingly high content of carbon and a high content of sulphur. As
a product of trypsin-proteolysis, it must presumably come from the
cleavage of hemipeptone, which, however, contains only 0.75 per cent.
of sulphur. But as we have seen, this latter body breaks down by
further cleavage into substances such as leucin, tyrosin, lysin, etc.,
which contain no sulphur whatever, and as there is no elimination
of sulphur in this process through formation of hydrogen sulphide
gas or otherwise (putrefaction being excluded by the presence of
either chloroform or thymol), it follows that this surplus sulphur
must accumulate somewhere. The high content of carbon, however, in
proteinochromogen is sufficient evidence that the substance cannot
have its origin in a simple cleavage of hemipeptone. On the other
hand, everything points to a synthetical process, in which two or more
cleavage products of the proteid molecule combine and form a new body,
such as proteinochromogen, containing all the sulphur cast off from the
hemipeptone in the production of the crystalline bodies, and having in
itself properties common to peptone and to a body of the indigo-group,
the latter obviously coming from some aromatic antecedent.
In view of the apparent complexity of the processes attending
trypsin-proteolysis, it is not strange that even simpler substances
than those already described should make their appearance. Thus, when
it was suggested that ammonia, NH_{3}, might be formed under the
influence of trypsin, it was not considered at all improbable, for in
the hydrolytic decomposition of proteids by boiling dilute acid, as
well as by baryta water, it had long been known as a prominent product.
Obviously, in trypsin-proteolysis, the one thing to be guarded against
in proving the formation of ammonia is the contaminating influence
of bacteria. Hirschler,[191] however, with a full recognition of this
danger, made digestions of blood-fibrin with trypsin extending only
through four hours and at a temperature of 32° C., and yet he obtained
plain evidence of the formation of ammonia. Stadelmann,[192] with
still greater precautions to exclude all bacterial agencies, using
boiled fibrin as the material to be digested and thymol to prevent
any possible infection of the digestive mixture, proved conclusively
that ammonia was formed as a result of trypsin-proteolysis. Thus, in
the digestion of 35 grammes of boiled blood-fibrin with 60 c. c. of
a pancreas infusion for three days, 20.8 milligrammes of NH_{3} were
developed, presumably coming from the liberation of a certain amount
of nitrogen attendant upon the formation of such bodies as leucin and
tyrosin, which contain considerably less nitrogen than their direct
antecedent hemipeptone, or the original proteid. We thus have striking
proof of the ability of this peculiar proteolytic enzyme to set in
motion hydrolytic changes which may extend even to the production of
such simple substances as ammonia, thus making still more striking
the parallelism between trypsin-proteolysis on the one hand, and the
artificial hydrolysis produced by boiling dilute acids on the other.
[191] Bildung von Ammoniak bei der Pancreasverdauung von Fibrin.
Zeitschr. f. physiol. Chem., Band 10, p. 302.
[192] Bildung von Ammoniak bei Pancreasverdauung von Fibrin. Zeitschr.
f. Biol., Band 24, p. 261.
In view of all these facts regarding the nature of the products
obtainable by pancreatic proteolysis, it is very evident that many
chemical changes may take place side by side in a vigorous pancreatic
digestion of proteid matter. We know without a shadow of doubt that all
of the bodies enumerated as products of pancreatic digestion are the
results of trypsin-proteolysis, and not the products of putrefactive
changes. Bacteria, it is true, are able to produce many like products,
and in the living intestinal tract exercise an important influence,
especially in the breaking down of resistant forms of proteid matter,
and in the decomposition of surplus material which has escaped the
pancreatic ferment. But all the bodies described above are readily
obtainable by trypsin-proteolysis under conditions which exclude all
possibility of bacterial action.
Granting, then, as we must, that these various bodies are all products
of pancreatic proteolysis when the process is carried on in beakers
or flasks, we need to consider next how far such bodies appear in
the natural process as it takes place in the living intestine. We
know indeed that the natural and the artificial processes are very
much alike so far as the qualitative results are concerned, but what
differences there may be between the quantitative relationships in the
two cases is less certain. One might naturally reason that, with the
facilities for rapid absorption that exist in the small intestine,
trypsin-proteolysis would rarely proceed beyond the peptone stage,
yet we have ample evidence that, under some circumstances at least,
both leucin and tyrosin are formed in considerable quantities in the
intestine.
It obviously makes a very great difference to the economy in what
form the proteid matter ingested leaves the intestine on its way into
the blood-current. It has been more or less generally assumed that,
under the ordinary circumstances existent in the intestinal tract, the
crystalline and other bodies coming from the more profound changes
incidental to trypsin-digestion are rarely formed, mainly on the
ground that such transformations would entail great loss of nutritive
material to the blood. Years ago, Schmidt-Mülheim[193] made a series
of experiments on the changes which proteid foods undergo in different
portions of the alimentary tract, from which he concluded that leucin
and tyrosin are formed in such small quantities in natural pancreatic
digestion that they represent only a very small part of the nitrogen
absorbed from the intestine. This conclusion has been more or less
generally accepted, especially as several observers have reported
finding only small amounts of these bodies in the intestine under what
might be assumed to be favorable circumstances for their formation. In
artificial digestions, on the other hand, as we have seen, leucin and
tyrosin, together with the other simple bodies described, may appear
in large quantities. Obviously, two suggestions present themselves as
explanatory of this difference; either there is such a rapid absorption
of these crystalline products from the intestine that they cannot be
detected other than as mere traces, or else the natural process takes
a different course from the artificial, owing to the rapid withdrawal
from the intestine of the antecedent of the leucin and tyrosin, viz.,
the hemipeptone.
[193] Untersuchungen über die Verdauung der Eiweisskörper. Du
Bois-Reymond’s Archiv f. Physiol., 1879, p. 39.
Concerning this point, Lea[194] has recently reported some experimental
evidence obtained by a comparative study of artificial pancreatic
digestion as carried on in a flask, with similar digestions carried on
in parchment dialyzer tubes, the latter so arranged that the diffusible
products of proteolysis can pass from the tube into the surrounding
fluid. As Lea justly says, this whole question of the formation
of leucin by proteolysis is a very important one, since it bears
closely upon one of the possible methods by which urea may be quickly
formed from proteid food. Thus, we have evidence that when leucin is
administered to mammals a portion of its nitrogen, at least, quickly
reappears as urea and uric acid in the urine.[195] Further, there is
a certain amount of evidence that this transformation takes place in
the liver, viz., in the organ where leucin absorbed from the intestine
would naturally be first carried.[196]
[194] A Comparative Study of Artificial and Natural Digestions. Journal
of Physiology, vol. xi, p. 226.
[195] E. Salkowski: Weitere Beiträge zur Theorie der Harnstoffbildung.
Zeitschr. f. Physiol. Chem., Band 4, pp. 55 and 100.
[196] W. Salomon: Ueber die Vertheilung der Ammoniaksalze im
thierischen Organismus und über den Ort der Harnstoffbildung. Virchow’s
Archiv, Band 97, p. 149.
Obviously, the main point to be gained in a dialyzer-experiment is
the removal of the soluble products of digestion as soon as they are
formed; but peptones are not rapidly diffusible, and the process,
as noted under the head of gastric digestion, cannot be considered
in any sense as yielding the same results as might be obtained in
the living intestine. Still, the method offers a closer approach
to the natural process than when carried on in a flask, and the
results are of interest. Thus, Lea finds in the first place that
in a dialyzer-digestion the proteid is more quickly dissolved, and
that there is far less tendency for the formation of an insoluble
antialbumid with its natural resistance to the ferment. Still, it is
to be noticed that the amount of this antialbumid-residue formed by
trypsin-proteolysis in a flask is mainly dependent upon the strength
of the ferment solution, and the character of the proteid undergoing
digestion. If the latter is in a fairly digestible form, and the enzyme
solution reasonably active, then even the flask-digestion may show
almost no residue of antialbumid. Yet there is at least a shade of
difference in the two cases, which may be expressed by the statement
that trypsin-proteolysis, as carried on in a dialyzer-tube, is prone
to give less insoluble antialbumid than a corresponding digestion
in a flask. Further, the amount of leucin and tyrosin formed in a
flask-digestion is always greater than in a dialyzer-digestion, other
conditions being equal. Naturally, these results help us very little
in drawing any conclusions regarding the extent to which leucin and
tyrosin may be formed in the intestine. They merely emphasize the
fact that the withdrawal of a certain quantity of hemipeptone from
the digestive mixture tends to reduce by so much the yield of leucin
and tyrosin. It is hardly to be assumed, however, that the rate of
withdrawal of peptone from the intestine can keep pace with its
formation, especially when it is remembered that the proteid matter
coming into the small intestine, owing to its preliminary treatment
in the stomach, is in a comparatively digestible condition. Further,
the pancreatic juice is a remarkably active fluid, and proteolysis
under its influence must make rapid strides. I can easily conceive
that proteolysis by trypsin, when carried on in a flask, may lead to
the formation of much larger amounts of leucin and tyrosin, and of
other bodies as well, than occurs in the natural process; but there is
certainly no ground for the belief that leucin and tyrosin are wholly
wanting in pancreatic proteolysis as it occurs in the intestine.
With a view to obtaining some positive evidence on this point I have
tried a few experiments on animals, the results of which have convinced
me that, in the case of dogs, at least, both leucin and tyrosin may
be formed in natural pancreatic digestion in considerable quantities.
Thus, in one experiment a good sized dog, kept without food for two
days, was fed four hundred grammes of chopped lean beef at 8 A.M.
At 2 P.M. the animal was killed and the intestine ligatured close
to the pylorus. The lower end of the small intestine was likewise
ligatured. The portion inclosed between the two ligatures was then
removed from the body, and the contents of the intestine pressed and
rinsed out with distilled water. In the stomach, was found a small
amount of semi-digested matter weighing about fifty grammes. The
material obtained from the intestine was strained through mull, the
fluid rendered faintly acid with acetic acid, and heated to boiling.
The clear filtrate from this precipitate was concentrated to a very
small volume, and while still hot precipitated with a large amount of
ninety-five per cent. alcohol. A small gummy precipitate resulted,
which was thoroughly extracted with boiling alcohol and the washings
added to the alcoholic filtrate. The precipitate contained some
deuteroproteose and a small amount of true peptone.
The alcoholic fluids were evaporated to a small volume and set aside
in a cool place. As a result, quite a separation of leucin and tyrosin
occurred in the characteristic crystalline forms. No attempt was
made to effect a quantitative separation of the two bodies, but the
mixed precipitate finally obtained weighed, after recrystallization,
over three-fourths of a gramme. Leucin was plainly in excess, but
considerable tyrosin must have been left in the alcoholic precipitate,
owing to its greater insolubility in this menstruum. This experiment
is almost a counterpart of one reported by Lea,[197] and like
his indicates that both leucin and tyrosin may be formed in not
inconsiderable quantities by pancreatic proteolysis as it occurs in
the intestine. This being so, one is naturally called on to explain
“the physiological significance of a process which at first sight
appears to result in a degradation of the potential energy of proteids,
under conditions such that the energy set free can be of little use
to the economy.”[198] But it is quite possible, as Lea has suggested,
that these amido-bodies have an important part to play in some of
the synthetical or other processes of the organism, and that their
formation is consequently necessary for the well-being of the body.
Whether this is so or not, we may well consider the formation of these
amido-acids in pancreatic proteolysis as a means of quickly ridding the
body of any excess of ingested proteid food, with the least possible
expenditure of energy on the part of the system. This has always seemed
to me the probable purpose of the profound changes which the pancreatic
ferment is capable of inducing.
[197] Journal of Physiology, vol. xi, p. 255.
[198] Lea: loc. cit.
The primary object of both gastric and pancreatic proteolysis is to
render the proteid foods more easily available for the needs of the
economy, viz., to aid in their absorption and consequent distribution
to the master tissues and organs of the body. This is doubtless
fully accomplished by the formation of the so-called primary and
secondary products of proteolysis, _i. e._, the proteoses and peptones
which are, comparatively, not far removed from the mother-proteid,
except in solubility and other minor points. In the ferment trypsin,
however, we have a special agent endowed with the power of carrying
on the hydrolytic cleavage to a point where exceedingly simple bodies
result, and through whose agency any excess of proteid material in the
intestinal canal may be quickly broken down into a row of products
easily removed from the system. It is to be remembered, however, that
the very nature of the proteid molecule precludes the possibility
of anything like a complete decomposition into crystalline or other
simple products. Full fifty per cent. of the peptone formed must
be antipeptone, which cannot be further changed by trypsin under
any circumstances, so that, whether the amount of proteid in the
intestine be large or small, or whether it is exposed for a longer or
shorter period to trypsin-proteolysis, there will always be a fairly
large amount for absorption. This may well be considered as one of
the reasons for the peculiar structure of the proteid molecule, the
anti-group being always available for the direct nutrition of the body,
while the representatives of the hemi-group, especially when proteid is
present in excess, can be quickly and readily broken down into simple
products. In other words, the direct formation of these simple bodies
in the intestine furnishes a short path to urea, thus leading to the
rapid elimination of any excess of proteid material.
We may well attribute to the epithelial cells of the intestine the
power, under normal circumstances, of regulating and controlling, even
though indirectly, the order of events in the intestine. Just as the
so-called secreting cells of the _tubuli uriniferi_ may lose for a time
their power to pick out from the blood material destined for the urine,
being clogged or exhausted by continued effort, so the epithelial cells
of the intestine, which play such an important part in the absorption
of proteid matters from the alimentary tract, may, in the presence
of an excess of proteid matter, become temporarily exhausted, and,
refusing passage to the proteoses and peptone formed by proteolysis,
render possible further hydrolytic cleavage into leucin, tyrosin,
lysatin, etc.; bodies which, by one method or another, can be readily
transformed into urea. At the same time, as already stated, it seems
more than probable that some formation of these amido-acids always
occurs in the intestine, and that these bodies have some specific part
to play in the normal processes of metabolism going on in the body.
The more one studies the processes of nutrition in general, the more
one is impressed with the view that there is a purpose in everything,
and that the formation of even such bodies as leucin and tyrosin may
be connected with hidden processes, the key to which has not yet
been found. We see an analogous case, perhaps, in the action of the
inorganic salts in nutrition, some of which, at least, neither undergo
change themselves nor induce changes in other substances, and yet we
know their presence is indispensable for keeping up the normal rhythm
of the nutritional processes of the body.
ABSORPTION OF THE MAIN PRODUCTS OF PROTEOLYSIS.
In ordinary proteolytic action, both in the stomach and intestine,
it is very apparent that the primary products of proteolysis, the
proteoses and peptones, are the chief products formed, and that under
normal circumstances the greater portion of the proteid food finds its
way from the alimentary canal into the blood, after transformation
into one or more of these two classes of products. At the same
time, it must be borne in mind that even the acid-albumin formed by
pepsin-hydrochloric acid may be absorbed without undergoing further
change. The view once held, that the rate of absorption from the
alimentary tract stands in close relation to the diffusibility of the
products formed, and that non-diffusible substances are incapable of
absorption, is no longer tenable. Absorption from the intestine is to
be considered rather as a process involving the vital activity of the
epithelial cells of the intestinal mucous membrane, where chemical
affinities and other like factors play an important part in determining
the rate and order of transference through the intestinal walls into
the blood and lymph. Thus, we have abundant evidence that native
proteids which have not undergone proteolysis may be absorbed from
the intestine, at least to a certain extent, provided they have been
dissolved; _i. e._, converted into acid-albumin, or alkali-albuminate,
by the gastric or pancreatic juice. We have a practical demonstration
of this possibility in the early experiments of Voit and Bauer,[199] as
well as in many later ones that need not be mentioned here. Further,
the recent experiments of Huber[200] have given us quantitative data
on the rate of absorption of fluid egg-albumin when introduced into
the large intestine in the form of a clyster, showing that even fairly
large amounts of a natural proteid may be absorbed without undergoing
proteolysis if mixed with a neutral salt, like sodium chloride. To be
sure, the rate of absorption is greatly increased when the albumin has
been peptonized, but still absorption of the native proteid is possible
without the agency of proteolytic enzymes. When, however, large amounts
of egg-albumin are introduced into the intestine, albuminuria may
result, as you very well know.
[199] Ueber die Aufsaugung im Dick und Dünndarm, Zeitschr. f. Biol.,
Band 5, p. 562.
[200] Ueber den Nährwerth der Eierklystiere, Arch. f. klin. Med., Band
47, p. 495.
Moreover, it is well known that the proteids of muscle-tissue, in the
form of syntonin, may be absorbed from the large intestine without
undergoing further hydration. When introduced into the rectum of a
hungry dog, the excretion of urea may be at once increased and the
animal brought into a condition of nitrogenous equilibrium; absorption
taking place from a portion of the large intestine, where proteolysis
is never known to occur.[201]
[201] Eichhorst: Ueber die Resorption der Albuminate im Dickdarm,
Pflüger’s Archiv f. Physiol., Band 4, p. 570.
Again, Neumeister[202] has shown that the direct introduction of
syntonin, alkali-albuminate, crystalline phytovitellin, as well as
pure serum-albumin, into the blood of the jugular vein is not attended
with the appearance of albumin in the urine. On the contrary, the
proteid matter so introduced appears to be assimilated and utilized
for the needs of the organism. Evidently, then, these substances are
not to be considered as foreign bodies, for if so the kidneys would
undoubtedly make some effort to remove them from the circulation. It is
to be noted, however, that all native proteids are not assimilated in
this manner, as casein,[203] gelatin,[204] and especially egg-albumin.
Thus, J. C. Lehman,[205] working under Kühne’s direction, observed
that the injection of a carefully filtered solution of egg-albumin into
the veins of a dog was always accompanied by albuminuria, while similar
injections of Lieberkühn’s sodium albuminate, or of syntonin from
frog’s muscle, failed to show any such result.
[202] Zur Physiologie der Eiweissresorption und zur Lehre von den
Peptonen, Zeitschr. f. Biol., Band 27, p. 309.
[203] Neumeister: Sitzungsber. der Physik. med. Gesellsch. zu Würzburg,
1889, p. 73.
[204] F. Klug: Pflüger’s Archiv f. Physiol., Band 48, p. 122.
[205] Virchow’s Archiv, Band 30, p. 593.
While these observations tend to show that some native proteids may
be absorbed from the alimentary tract without previously undergoing
proteolysis, it is not to be understood that any considerable quantity
is so absorbed under normal circumstances. Doubtless, when small
amounts of proteid food are taken, its denaturalization by the primary
action of the gastric or pancreatic juice, viz., its conversion into
syntonin or alkali-albuminate, may be sufficient to insure its partial
absorption, but digestive proteolysis is unquestionably a necessary
preliminary to any general absorption, and there can be no manner of
doubt that the greater portion of the proteid food is absorbed as
proteoses and peptone. Peptones, as we have seen, are possessed of
a higher endosmotic equivalent than the proteoses, but we need to
keep continually in mind the possibility that the selective power of
the epithelial cells of the intestinal mucosa may lead to as rapid
transference of the proteoses as of the more diffusible peptones. It
is not to be understood by this, however, that diffusibility is of no
consequence in determining the rate of absorption. Surely, everything
else being equal, the more diffusible the substance the more rapid will
be its passage from the intestine into the blood-current. The more
the process of absorption is studied, however, the more clearly do we
see its dependence upon the functional power of the living epithelial
cells, a fact which plainly emphasizes the physiological nature of the
process.
Further, as already stated, absorption of proteid food-stuffs, or their
products, from the alimentary tract, is, under ordinary circumstances
at least, limited to the intestine; from the stomach there is
comparatively little absorbed, and if necessary we might advance this
fact as an important argument against the theory of general absorption
of proteids in the form of acid-albumin. Even such indifferent fluids
as water, or physiological salt solution, are absorbed with extreme
slowness from the stomach;[206] this organ showing very little ability
to take up water even when the blood-vessels are dilated, as after the
ingestion of food.
[206] J. S. Edkins: The Absorption of Water in the Alimentary Canal.
Journal of Physiol., vol. 13, p. 445.