E. A. (Edward Albert) Sharpey-Schäfer.

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watery and saline constituents of the lymph will be reabsorbed until the
original constitution of the lymph is restored.

We may conclude, therefore, in default of definite evidence to the
contrary, that while the interchange between tissues and blood, so far as
diffusible substances are concerned, is effected by diffusion through the
medium of the lymph, the proteid supply to the cells is dependent on
the amount of proteid transuding with the lymph.

Perhaps it is on this account — i.e., increased proteid supply to the
cells — that chronic inflammation or hypersemia of any part is apt to lead
to its hypertrophy. . Growing tissues, as well as those in a state of repair,
have delicate vessels, which probably supply a lymph much richer in
proteids than is supplied to adult tissues.


By B. Mooee.

Contents : — Digestive Ferments, p. 312 — Chemical Composition of Digestive Juices,
p. 342 — Saliva, p. 342 — Gastric Juice, p. 349 — Pancreatic Juice, p. 366 — Intes-
tinal Juice, -p. 368— Bile, p. 369 — Digestion of Carbohydrates, -p. 392 — Digestion
of Proteids, p. 428 — Absor2:)tion of Carbohydrates and Proteids, p. 430 —
Digestion and Absorption of Fats, p. 443 — Bacterial Digestion, p. 463 — Com-
l^osition of Faeces, p. 472.

The Digestive Ferments, or Enzymes.

Organised and unorganised ferments. — Fermentation is invariably
brought about, directly or indirectly, by cell life, either vegetable or
animal. When the action is direct, and the chemical changes involved
in the process occur only in the presence of the cell, the latter is
spoken of as an organised ferment. When the action is indirect, and
the changes are the result of the presence of a soluble material secreted
by the cell acting apart from the cell, this soluble substance is termed
an unorganised ferment, solvMe ferment, or enzyme}

The action of an organised ferment is intimately connected with the
life of the cell, and is instantly stopped by anything which either kills
the cell or temporarily arrests its activity; while that of a soluble
ferment is not a vital process, but one which is purely physical or
chemical in its nature. As a consequence, an organised ferment is
destroyed, and its specific action stopped, by any protoplasmic poison,''^
while an unorganised ferment, provided it is not precipitated, is un-
affected by such reagents.

All the differences m the mode of action of organised and un-
organised ferments arise from this close connection of the organised
ferment with the cell. Thus, an organised ferment, provided there is a
supply of nitrogenous food at its disposal, can grow and multiply in a
medium in which it is sown, while an unorganised ferment can never so
increase in quantity ; from this it follows that the rapidity of action of
an unorganised ferment depends (within limits) on the initial quantity
added, but in the case of an organised ferment the initial amount soon
becomes a matter of no moment.

Organised ferments are unicellular organisms (microfungi), while the

^ This term was first used by Ktihne, Verhandl. d. naturh.-med. Ver. zu Heidelberg,
1879, N. F., Bd. i. S. 236.

^ Such as any of those substances commonl}'^ known as antiseptics.


unorganised ferments are typically found in the secretions of specialised
cells of the higher plants and animals, and take an important i^art in
the chemical changes involved in their nutrition.

There is probably at bottom very Httle difference in the manner of action
of cellular ferments and enzymes. From the cell substances of several
bacteria, extracts have been obtained possessing the same fermentative action
as the living bacteria ; this indicates that in such bacteria, substances are
present in the cell which act like ordinary unorganised ferments, but normally
remain during the life of the cell within its substance, and perform their
fermentative functions there.

A good example of such an isolation of an unorganised from an organised
ferment, is afforded by that series of brilliant researches into the nature of
the action of the micro-organism, torula urex, upon urine, which began with
the observation that the change into ammonium carbonate was not stopped by
the presence of carbolic acid in sufficient amount to paralyse the growth of
the micro-organism,! and ended in the extraction from the bacteria of
a soluble ferment, which converted urea into ammonia and carbonic acid, even
in the presence of chloroform, which effectually stops all bacterial action. ^

In a similar manner, a soluble ferment, capable of inverting cane sugar,
can be extracted from yeast cells after they have been killed by the action of
alcohol or ether,^ and from certain putrefactive bacteria unorganised ferments
have been obtained, possessing an action on proteids analogous to that of the
proteolytic ferment of the pancreatic juice. Such intracellular soluble ferments
have not been shown to exist in by far the greater ni;mber of organised ferments,
but if they do so exist the only remaining difference between organised and
unorganised ferments is that in the former the substance formed by the cell
remains in the cell substance, and does its work there, the products of its
action being poured forth as a kind of secretion or excretion, while in the
latter the ferment becomes separated from the cell in a secretion, and carries
out its work apart from the cell.

Most of the chemical changes involved in the digestion of the food
are brought about by the presence in the digestive secretions of soluble
ferments. So that digestion might he described as the ijhysical and chemical
alteration of the foodstuffs, into forms tetter fitted for absorption, by the
action of certain soluble ferments, the digestive enzymes.

Attempts to isolate pure enzymes. — Many attempts have been
made to isolate chemically pure enzymes, but the task is very diiScult,
and it is highly probable that no one has yet succeeded in obtaining a
pure product.

There are two great difficulties in the way : first, our ignorance of a
specific precipitant for any of the enzymes ; and, secondly, the extremely
small quantities in which they are present in the secretions. On account
of the first, the enzyme cannot be thrown out of solution unaccompanied
by other substances ; on account of the second, it is not present in
workable quantity, and is rapidly lost in any lengthened process of
chemical manipulation. When to these disadvantages are added the
non-diffusibility of the enzymes, which shuts out a means of separating
them from the traces of proteid which always accompany them, and
their sensitiveness to reaction and temperature, some idea is obtained of
the difficulties which the problem of isolation presents.

^ Hoppe-Seyler, Med.-chem. Untersucli., Berlin, 1871, Heft 4, S. 570.

^ Sheridan Lea, Joiorn. Physiol., Cambridge and London, 1885, vol. vi. p. 136.

^ Hoppe-Seyler, Ber. d. deutscli. cliem. Gesellsch., Berlin, 1871, S. 810.


Method of mechanical iwecipitation. — When an indifferent precipitate
is produced in a solution containing an enzyme, this is often carried
out of solution with the precipitate, probably in a condition of mechanical
adhesion. This observation was made by Briicke, who utilised the
property to free pepsin as far as possible from other substances. The
method has been extended to the preparation of purified forms of other
enzymes, and, as applied by Briicke ^ to pepsin, may be quoted as an
example of a general method. It is as follows : —

The mucous membrane of a pig's stomach is submitted to partial self-
digestion, in water acidulated with phosphoric acid ; the products of this
first digestion are rejected, being too rich in products of digestion, and not
containing much pepsin, which clings in great part to the mucous membrane.
The residue of the mucous membrane is again digested in water made acid
with phosphoric acid, and after some days is filtered from insohible residue,
and just neutrahsed by the addition of hme water. The insohible calcium
phosphate so precipitated carries down with it all the pepsin ; it is collected on
a filter paper, just dissolved by cautious addition of very dilute hydrochloric
acid, filtered off", and once more precipitated by the addition of just sufficient
lime water. This double precipitation is to free the pepsin of proteid, which
also has the property of being mechanically carried down, though more feebly
than pepsin. To this somewhat purified solution of pepsin a solution of
cholesterin in four parts of alcohol and one part of ether is added. On this
solution mixing with the water the cholesterin becomes insoluble, and is thrown
out of solution in a finely divided condition, carrying the pepsin mechanically
adhering to it just as it did to the calcium phosphate. The mixture is well
shaken up, ancl then filtered ; the precipitate is washed first with water, then
with water acidulated with acetic acid, and finally with water alone. It is
next, without drying, shaken up with ether, free of alcohol, but saturated
with water. The ether extracts the cholesterin, while the pepsin remains in
the watery layer beneath ; the extraction is repeated with fresh portions of
ether until all the cholesterin has been removed, and finally the watery
solution containing the pepsin is filtered. In this manner a solution is
obtained, which actively peptonises, but contains so little proteid as not to give
many of the proteid reactions.

Method of auto-digestion. — Klihne and Chittenden ^ have combined
auto-digestion with precipitation by ammonium sulphate as a means of
preparing purified solutions of pepsin and trypsin. The following is an
outline of their methods : —

For the preparation of pepsin the mucous membrane of a pig's stomach is
taken, and allowed to undergo auto-digestion for several days, until peptonisa-
tion has far advanced, and but comparatively little albumose is left. The
solution, after filtration from undigested debris of nuclein, etc., is next satu-
rated with ammonium sulphate. The pepsin is thus completely thrown out
of solution along with the albumoses ; this precipitate is dissolved again, after
pressing in filter paper, in dilute hydrochloric acid, and allowed to go on
finishing the digestion of the albumoses for some days.

The process is repeated as often as is necessary to remove the albumose,
and finally the pepsin, after being dissolved by addition of water, is freed from

1 Sitzungsh. d. k. Akad. d. Wissensch. , Wien, 1862, Bd. xliii. S. 601 ; " Vorlesungen ii.
Physiologic," Wien, 1885, S. 308. See also v. Hcltzl, Jahrcsb. it. d. Fortschr. d. ges. Med.,
Erlangen, 1864, Bd. i. S. 138.

" Ztschr. f. Biol., Mlincbeii, 1886, Bd. xxii. S. 428. Suoli a method of auto-digestiou
can obviously only be employed in the case of proteolytic enzymes.


ammonium sulphate by dialysis, and may be precipitated by alcohol, filtered
off and dried as quickly as possible.

The preparation of a purified trypsin solution is carried out by
Klihne's ^ method much on the same lines : —

The pancreas first has all its fat removed by extraction with alcohol
followed by ether, after which process it forms Klihne's "pancreas powder."
This is digested with five times its volume of 0"1 per cent, salicylic acid for
about four hours. The residue is next digested with 0*25 per cent, sodium
carbonate solution for a further period of twelve hours, and the solution is
separated from the undissolved part. The two extracts are noAV mixed, the
mixture made up with carbonate of sodium solution to a strength of 0*25 to a
0*5 per cent, carbonate, and allowed to digest at 40° C. for a week, thymol being
added to prevent putrefaction (0'5 per cent.). During this time the albumoses
become converted into peptones, and on saturating the cold solution, made
very faintly acid with acetic acid, with ammonium sulphate, trypsin is
precipitated, accompanied by traces only of unconverted albimioses. The pre-
cipitate so obtained is sufficiently pure for all digestion experiments. It
contains so little accompanying albumose, that, from 10 grms. of pancreas
powder, merely a thin yellowish slime is obtained on the filter paper, yet this,
when taken up by 100 c.c. of 0"25 per cent, sodium carbonate solution, forms
a strong digestive fluid. This gives an idea of the extreme power of the
digestive ferments, and shows at the same time in what mere traces they
must be present in the glands. This product may be still further purified by
partially precipitating the solution obtained from it with excess of alcohol,
dissolving in water, separating by dialysis the bulk of the ammonium sulphate
also precipitated by the alcohol, removing the last traces of ammonium sulphate
by barium carbonate, and finally precipitating as a snow-white amorphous
substance by excess of alcohol.

This pure product gives all the proteid reactions (unlike Brlicke's
pepsin), but in spite of all the elaborate and painstaking processes used
in its preparation, there is no evidence that it does not still contain
traces of proteid along with trypsin; the other conclusion of course
would be that trypsin is itself a proteid.

Preparation of digestive extracts. — When the object is simply to
test or demonstrate the action of the enzymes, and the admixture of
products of digestion formed from the gland tissue is a matter of no
moment, much simpler methods of preparation may be employed than
those above described.

1. In many such cases a simple extraction of the gland Avith water may
be used, if the action is to be tested immediately.

2. A general method of obtaining digestive extracts is that first recom-
mended by V. Wittich,^ which consists in preparing a glycerin extract. Such
an extract has the advantage of efficiency and stability. It contains a good
deal of proteid, and cannot be used where the products of digestion are to be
exactly studied, but for general laboratory work glycerin extracts are most
convenient preparations. They are easily made, and may be preserved for
years. As the glycerin only slowly extracts the enzymes, the same tissue will
continue for a long time to yield fresh extracts, if fresh glycerin be added.

A glycerin extract should not be made with a quite fresh gland, but with

^ JlnUrsuch. a. d. physiol. Inst. d. Univ. Heidelberg, 1878, Bd. i. S. 222 ; Verliandl. d.
naturh.-med. Ver. zu Heidelberg, 1886, N. F., Bd. iii. S. 463. See also ibid., 1876, N. F.,
Bd. i. S. 195.

2 Arch.f. d. ges. Physiol., Bonn, 1869, Bd. ii. S. 193; 1870, Bd. iii. S. 339.


a gland which has been minced up and allowed to stand for a few hours (it
may be in nearly all cases made faintly acid with very dilute acetic acid to set
free the zymogen as enzyme). Such a minced-up gland is rubbed up in a
mortar with some clean sand, taken up with glycerin, shaken up with more
glycerin (10-20 parts to 1 part of gland), and allowed to stand so until
required ; the process of extraction is very slow, and requires from seven to
fourteen days. In the case of gastric mucous membrane, 1 part per 1000 of
hydrochloric acid may be added to the glycerin.

There are many modifications of the process. v. Wittich recommends
digesting the minced gland (or mucous membrane) twenty-four hours in alcohol,
drying after this in the air, sifting the powder through gauze to remove
coarser fragments of tissue, and extracting with glycerin. It is often recom-
mended to filter the extract after seven to fourteen days, but this is unnecessary,
as the tissue neither decomposes nor becomes digested in the glycerin, and
the extract improves on keeping in contact with the tissue. The enzyme
accompanied by proteid may be precipitated from a glycerin extract by the
addition of absolute alcohol, and so a purer extract be obtained.

Chemical nature of enzymes. — The failure of all attempts to isolate
pure enzymes necessarily deprives us of the possession of any certain
knowledge of the chemical nature of these substances. Analyses of the
purer preparations of the enzymes give figures approximating to those
obtained with the various proteids ; but whether or not this is due to
admixture with proteid it is at present impossible to say. The behaviour
of Briicke's " pure " pepsin solution goes against the supposition that this
enzyme is a proteid. This solution did not give the proteid reactions,
and was not precipitated by any of the proteid precipitants, save neutral
and basic lead acetates and platinic chloride. These results are confirmed
by Sundberg,^ who succeeded in preparing a still more proteid-free solu-
tion, which did not even react to these reagents, and was only precipi-
tated as a slight, pure white, flocculent precipitate, on adding five to six
times its volume of absolute alcohol and allowing to stand, and yet was
exceedingly active in digesting fibrin. The amount of this precipitate
was much too small for analysis, and it could only be shown that it was
nitrogenous, and contained a certain amount of ash. This is not quite
conclusive against the proteid nature of the active substance, since, as
Sundberg argues, the physiological test by digestion may be much more
delicate than any of the purely chemical tests. Still, the fact that it
was totally unaffected hy tannic acid and precipitated by alcohol has
some weight against the substance being proteid in nature ; since tannic
acid will show 1 part of ordinary proteid in 100,000,^ and alcohol is by
no means so dehcate a proteid test. It is most probable, then, that
pepsin is not a proteid ; and it will subsequently be seen in the descrip-
tion of the other enzymes that most of these have been obtained in
forms which do not yield all the proteid reactions.

The enzymes are soluble in water, from which they are precipitable
by saturation with ammonium sulphate or by adding excess of alcohol.^
Most of them are unalterable, or very slowly alterable in contact with
alcohol, but pepsin is an exception, being attacked and rendered inactive
if left long in contact. The enzymes are commonly said to be soluble

1 Ztsehr. f. 'pliysiol. Cliem., Strassburg, 1885, Bd. ix. S. 319. See also under "Ptyalin."
" Hofmeister, Ztsehr. f. phydol. Chem., Strassburg, 1878-9, Bd. ii. S. 292.
^ These may only be particular cases of theii' general mechanical precipitation, whenever
a precipitate is caused.


in glycerin, but it has not been shown that the dried enzymes are
soluble in anhydrous glycerin ; on the contrary, Kiihne ^ states that
pure trypsin is not soluble in strong glycerin ; and it is well known
that, after precipitation by alcohol from glycerin extracts, the enzymes
are afterwards much less soluble in glycerin.^

An elevated temperature rapidly destroys all enzymes when in
solution, and it is of some importance that the temperature at which
they are rapidly destroyed, although it varies considerably with the
reaction of the solution, lies just a little below the range at which the
bulk of the proteids coagulate. In the dried condition the enzymes are
much more resistant to increased temperature, and can be heated to over
100° C. for some time without losing their digestive properties on cooling
and dissolving in water.

The digestive action of the enzymes is not stopped by the presence
of disinfectants, such as thymol, chloroform, or salicylic acid, in quantity
sufficient to stop completely the action of organised ferments, particu-
larly that of the putrefactive bacteria.^ This fact has been turned to
account practically in conducting prolonged digestion experiments,
especially when the digestive action must be allowed to proceed in
alkaline solution.

Mode of action of enzymes. — The manner in which ferments bring
about the clianges characteristic of them is very puzzling. The enzymes
are altogether unaffected by the changes which they occasion, and, pro-
vided the products of the action are not allowed to become concen-
trated in solution, the ferment can work on indefinitely, and a finite
amount of ferment can convert an infinite amount of material. The
ferment may become by dilution, or unavoidable loss in manipulation,
so weak that finally its action becomes inappreciable ; but before this
happens it can be shown that it has converted a mass of material so
many times greater than its own, that the idea that it undergoes any
permanent alteration in the reaction which it induces must be abandoned.
Thus, according to Hammarsten,* one part of rennin will curdle 400,000
to 800,000 parts of milk ; while Petit ^ prepared a pepsin powder which
in seven hours dissolved 500,000 times its weight of fibrin.

There are numberless examples of chemical reactions, in which only
well-known and much simpler compounds take a part, of a substance
inducing a chemical reaction without itself becoming altered thereby.
Such a substance is called a catalytic agent, and the reaction a catalysis
or catalytic reaction. Ferment actions are such catalytic reactions, but
when we say that ferments act catalytically the problem of how they
act is not by any means solved ; we have merely found a name for it.

In some cases, in which the presence of a substance is essential to a
certain reaction, although this substance is not finally altered thereby,
there is evidence that it is altered intermediately and rechanged again
back to its initial condition during the reaction.

Such a case is to be found in the action of sulphuric acid in the con-
tinuous etherification process for producing ether from alcohol. It can
be shown that the sulphuric acid first combines with part of the alcohol

1 Verhandl. d. naturli.-med. Ver. zu Heidelberg, 1876, N. F., Bd. i. S. 196.

2v. Wittich, Arch.f. d. ges. Physiol., Bonn, 1869, Bd. ii. S. 193.

^ Kiihne, Verhandl. d. naiurh.-med. Ver. zu Heidelberg, 1876, IST. ¥., Bd. i. S. 190.

^ Jahresb. il. d. Fortschr. d. Thier-Chem., Wiesbaden, 1877, Bd, vii. S. 166.

^ Journ. de thdraj)., Paris, 1880.


molecule, forming a substance which can be isolated, and is known as
ethylsulphovinic acid ; and that this compound then reacts with another
molecule of alcohol, forming ether and regenerating the sulphuric acid
molecule, which is then free to repeat the process, and can be made to
do so indefinitely.

This action may be represented thus : —

(1) C2H5.O.H + ^ SO4 = H.O.H + ^^"^ SO4

(alcohol, sulphuric acid) (water, ethylsulphovinic


(2) ^^H^ SO, + C,H5.0H = ^80,+ ^^

(ethylsulpho- (alcohol) (sulphuric (ether)

vinic acid) acid)

Another good example of such an interaction is that of the alternate
formation of a higher oxide of nitrogen (NgOg) from a lower (NO), and
then the regeneration of the lower oxide, which is said to occur in the
formation of English sulphuric acid; the oxygen taken up in each
cycle going to form, with sulphur dioxide and water, sulphuric acid ;
while, as a net result, the nitric oxide remains unchanged, and may take
action again and again until it is dissipated by diffusion or otherwise.^

Such a part the enzyme may take in a ferment action ; a molecule
of it may unite with a molecule of the substance undergoing digestion.
Thus an unstable compound may be formed ; the elements of a water
molecule may combine with those of the fermentable substance, forming
a new substance ; while the ferment is regenerated to undergo another
cycle. Of all this, however, there is no experimental evidence ; there
is only the analogy, and analogies are sometimes misleading.

Besides these reactions, there are others in which the action of the
catalytic agent is, almost undoubtedly, merely a physical one ; that is to
say, in which the catalytic agent does not combine with the catalysed
substance, and then become regenerated. Such an action, for example,
is that of a trace of iodine in converting amorphous into red
phosphorus. Here the amount of iodine required is too excessively
small to suppose that it combines with phosphorus in one form and
yields it up in the other. The supposition is more probable that the
iodine finds the phosphorus in an unstable state, and in some fashion
enables it to do that which it already has a tendency to do, namely, swing
into stability. Such a reaction, only still more physical in character,
is found in the case of exceedingly unstable compounds (such as
detonating substances), where mere mechanical percussion, most probably

Online LibraryE. A. (Edward Albert) Sharpey-SchäferText-book of physiology; (Volume v.1) → online text (page 44 of 147)