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of lead, and then extracted the precipitate with nitric acid ; the insoluble
part left behind, his resin of bile, was impure glycocholic acid. The filtrate
he reprecipitated with excess of acetate of lead, collected the precipitate,
and decomposed it by a current of sulphuretted hydrogen, thus obtaining
his picromel, which must have corresponded to impure taurocholic acid.

In 1826, Gmelin published a memoir ^ in which is described a large
number of bile constituents ; amongst them, one corresponding to
glycocholic acid, which he obtained in a crystalline form ; and another
substance, taurine, of which he was the discoverer, although he wrongly
supposed that it existed ready formed in the bile. The next important
advance was made by Demarcay,* who obtained a substance {Choleinsdure)
which yielded, on heating with acids, taurine and a resinous substance,

1 Ztschr.f. physiol. Chcm., Strasskirg, 1888, Bd. xii. S. 196. ^ Paijlaill, loc. cit.

^ " Die Verdauuiig nach Versuchen."

■* Ann. d. Chem., Leipzig, 1838, Bd. xxvii. S. 270.



THE BILE SALTS. 373

and, on treatment with alkalies, ammonia and a non-nitrogenous acid,
corresponding to what to-day is called cholalic acid.

In 1844, Plattner^ succeeded in obtaining the bile salts in a
crystalline form, and so laid a sure foundation for all succeeding work on
the isolation and study of the bile acids. He also showed, by boihng
this crystalline product with acid, that taurin is a decomposition product
and does not exist as such in bile. Eedtenbacher - previously to this had
shown that this body contains sulphur, and established its formula as
C2H-NSO3. Plattner^ afterwards discovered a simpler method of
obtaining the mixed bile salts in crystalline form. He concentrated the
bile without decolorising, and then added an excess of alcohol, warmed,
and after some time filtered and added ether, till a brown sticky
precipitate began to fall ; this was allowed to settle, and the clear fluid
decanted off, cooled, and treated with more ether from time to time.
The bile salts alone being the only constituents which are soluble in
water and alcohol, and insoluble in ether, are slowly thrown out of
solution ; and on standing for some days or weeks in the cold, under the
alcoholic ethereal mother-liquid, form themselves into ball-shaped masses,
or starlike clusters of fine needles, which increase in size on standing.
This crystalline mass is known as " Plattner's crystallised hile." The
crystals are dried between filter-paper, washed with alcohol, containing 1
in 10 of ether, purified by recrystallisation, and dried over sulphuric acid.

This discovery of Plattner's paved the way for the classical researches
of Strecker, to whom we owe the greater part of any exact knowledge we
have of the bile acids. Strecker * first showed that " Plattners crystallised
hile " consists of a mixture of the sodium salts of two acids, which are so
related to each other that they yield, on boiling with acids, a common
non-nitrogenous constituent, cholalic acid, and a nitrogenous constituent,
which in both cases is an amido-acid. One of these amido-acids is
glycocoU or amidoacetic-acid, the other taurine or amidoethylsulphonic-
acid. Of the two bile acids the one which yields glycocoU and cholalic
acid is called glycocholic acid, while the other, which yields taurine and
cholalic acid, is named taurocholic acid.

Cholic or cholalic acid is not, however, the only basis of the different
varieties of bile acids ; other acids closely allied to it in percentage com-
position, but quite distinct from it, have been isolated. In ox bile about
a third part of the cholahc acid is replaced by an acid called choleic
acid.^ In human bile an acid called f ellic acid ^ has been described as
occurring along with cholalic and choleic acids ; and modified cholahc
acids are present in the hyoglycocholic acid of pig's bile and the cheno-
taurocholic acid of goose bile. None of these substitutes of cholalic
acid occur free in bile, but always combined with glycocoU or taurine
to form modified glycocholic or taurocholic acids ; they are all soluble
with difficulty in water and ether, and easily soluble in alcohol.''

^Ann. d. Chem., Leipzig, 1844, Bd. li. S. 1,05. ^ Ibid., 1846, Bd. Ivii. S. 170.

^ Journ.f. 2^rald. Chem., Leipzig, 1847, Bd. xi. S. 129.

* Ann. d. Chem., Leipzig, 1848, Bd. Ixv. S. 1 ; 1848, Bd. Lxvii. S. 1 ; 1849, Bd. Ixx.
S. 149.

^ Latscliinoff, JBer. d. deictsch. chem. Gesellsch., Berlin, 1885, Bd. xviii. S. 3039 ; 1886,
Bd. xix. S. 1140 ; 1887, Bd. xx. S. 1043.

® Fellinsdure of Schotten, Ztschr. f. physiol. Chem., Strassbnrg, 1887, Bd. xi. S. 268. See
also Lassar-Cohn, Ber. d,. deutsch. chem. Gesellsch., Berlin, 1894, Bd. xxvii. S. 1339.

■^ Hammarsten's "Lehrbnch," 1895, S. 198. He descaibes a third variety of bile acid,
found in shark's bile, which is rich in sulphur, and from which boiling with hydrochloric
acid splits off sulphuric acid.



3 7 4 CHEMISTR Y OF THE DIGESTIVE PR O CESSES.

The alkaline salts of the bile acids are soluble in water and alcohol,
but insoluble in ether, and these solubilities form the basis of Plattner's
method of separating them from the other biliary constituents. This
is best done by mixing the bile with freshly-heated animal charcoal,
evaporating to complete dryness, and then extracting with absolute
alcohol, which takes up the bile salts along with cholesterin and traces
of lecithin, fats, and soaps ; but, on addition of excess of ether, only the
bile salts are thrown out of solution.

The relative amount of each of the bile acids present in bile varies
within wide limits. In the bile of carnivora, glycocholate of sodium is
present in very small quantity ; for example, the bile salts of dog's bile
consist exclusively of taurocholate of sodium,^ while in most herbivora
the glycocholate is usually present in greater quantity than the tauro-
cholate ; to this rule the goat and sheep are said to be exceptions.

In human bile most of the cholahc acid is combined with glycocoll,
occasionally the whole of it.^ Hammarsten's ^ analysis of the mixed bile
salts of healthy human bile gave 13'1 per cent, taurocholic acid, 86 "9
per cent, glycocholic acid. Since glycochohc acid is sulphur-free, and
the percentage in taurocholic acid is known, the relative amount of the
two acids may be determined from the percentage of sulphur in a
preparation of Plattners crystallised tile, obtained from any given sample
of bile.

The isolation of each of the bile acids from a mixture of their
salts is usually a lengthy and difficult process, especially in the case of
taurochoUc acid, which can only with great difficulty be freed from
glycocholic acid, so that taurocholic acid is usually prepared from dog's
bile, while glycocholic acid is prepared from ox bile.

Both free acids behave like their sodium salts in being soluble in
alcohol and insoluble in ether, but differ in that taurochoHc acid is easily
soluble in water, while glycocholic acid is soluble with great difficulty.
On this property is based the simplest method of obtaining pure
glycocholic acid, that of Hiifner;* unfortunately, the presence of
taurocholic acid confers solubility on the glycocholic acid, so that the
method often fails when too much taurocholate is present in the sample
of bile experimented upon.

The method consists in adding to fresli ox bile a few drops of hydrochloric
acid, and filtering from the precipitated pseudo-mucin. To 100 c.c. of this
filtrate 5 c.c. of concentrated hydrochloric acid and 30 c.c. of ether are added.
The hydrochloric acid sets free both bile acids, and the glycocholic acid is
precipitated in crystalline form (unless too much taurocholic acid he present),
either immediately, or on standing some hours in the cold. The ether added
aids in the production of this crystalline precipitate, which is next washed
with acidulated water saturated with ether, and finally recrystallised from
boiling water.

Marshall^ tested Hiifner's method Avith 543 samples of ox bile, and
obtained a precipitation in 121 cases. A similar method Avas employed by
Strecker,*^ using a Avatery solution of crystallised bile instead of fresh bile.

^ Strecker, Ann. d. Chem., Leipzig, 1849, Bd. Ixx. S. 178; Hoppe-Seyler, Journ. f.
'pralct. Chem., Leipzig, 1863, Bd. Ixxxix. S. 283.

2 Jacobson, Ber. d. deutsch. chem. Gesellsch., Berlin, 1873, Bd. vi. S. 1028.

^ Schmidt's Jahri., Leipzig, 1879, Bd. clxxxi. S. 5.

^ Jahresi. 'it. d. Fortsehr. d. T/iier-Ohem., Wiesbaden, 1874, Bd. iv. S. 301.

^ Ztschr. f. 2)hysiol. Chem., Strassburg, 1887, Bd. xi. S. 233.

^ Ann. d. Cham., Leipzig, 1848, Bd. Ixv. S. 1.



THE BILE A CIDS. 3 7 5

The different solubilities of the lead salts of the two acids provides
another means of separating glycocholic acid; the separation of pure
taurocholic acid from the mixture by this method is more difficult.

Glycocholate of lead is thrown out of solution on the addition of neutral
acetate of lead to a solution of a mixture of the bile salts ; the remainder of
the glycocholate and all the taurocholate are thrown down on the addition of
ammonia or of basic acetate of lead to the filtrate.

Fresh ox bile is treated with alcohol to precipitate the pseudo-mucin.
The alcohol is evaporated off, and neutral acetate of lead added as long as a
precipitate forms ; this precipitate is collected and decomposed by warming
with a solution of sodium carbonate, whereby sodium glycocholate is formed ;
the mixture is next evaporated to dryness, and extracted with alcohol, in which
the sodium glycocholate dissolves. This alcoholic solution is filtered, the
filtrate is evaporated to dryness, and the residue is dissolved in water. The
watery solution of sodium glycocholate so obtained is decolorised with animal
charcoal, and the glycocholic acid thrown out of solution by adding a mineral
acid. Finally, it can be recrystallised, either from boiling water, or by the
addition of ether to its alcoholic solution. Taurocholic acid can be obtained
from the filtrate from neutral acetate of lead, by fractional precipitation with
basic acetate of lead, as the remaining glycocholate unprecipitated by the
neutral acetate is precipitated by the portion of basic acetate first added.^
Basic acetate of lead is stirred into the filtrate from the neutral acetate, until
the precipitate commences to gather into a sticky mass, when the addition is
discontinued, and the solution decanted off from the precipitate. More basic
acetate solution is now added, and throws down a plastic mass, consisting of
fairly pure taurocholate of lead. This precipitate is dissolved in boiling
alcohol, filtered warm into water, and the resulting reprecipitated mass, after
being purified by kneading, is dried, dissolved in a small quantity of alcohol,
decomposed with sulphuretted hydrogen, filtered from lead sulphide, and
dried at first in the air, afterwards in a vacuum over sulphuric acid.

Taurocholic acid is, however, best prepared from dog's bile, as
described by Parke.^

The bile is evaporated down, extracted Avith alcohol, decolorised with
animal charcoal, evaporated to dryness, dissolved in absolute alcohol, and
treated with excess of ether. After some time the crystalline precipitate of
sodium taurocholate so obtained is dissolved in water, and the solutions
precipitated with acetate of lead and ammonia. The precipitate is collected,
washed, suspended in alcohol, or dissolved therein by boiling, and decomposed
by sulphuretted hydrogen. The filtrate from sulphide of lead is evaporated
to a small volume, and mixed with excess of ether, when the taurocholic acid
is precipitated as a syrup, in which, after some time, small crystals appear.
These are in the form of fine needles which deliquesce in the air.

Glycocholic acid (C26H43NO6) is a monobasic acid, crystallising in
long fine needles, which fell together into a light, voluminous mass
when first formed from a solution, and on drying form a loose, snowy
white mass with a silky glance. These crystals melt at 100° C, losing
water in so doing and forming glycocholonic acid ; they are very
sparingly soluble in cold water (1 in 300), somewhat more soluble in
boiling water (1 in 120), and so can easily be recrystallised from hot
water ; they are easily soluble in alcohol and in acetic acid, but soluble
in ether with great difficulty. Glycocholic acid and its salts in solution

^ Lieberklilm, Jaliresh. il. d. Forfschr. d. ges. Med., Erlangen, 1852, Bd. i. S. 113.
^ Boppe-Seyler's 3fed.-chem. Untcrsuch., Berlin, S. 160.



376 CHEMISTRY OF THE DIGESTIVE PROCESSES.

rotate the plane of polarised light to the right ; in alcoholic solution the
specific rotatory power for the acid is +20°'0, for the sodium salt -f 25°"7
(Hoppe-Seyler). The salts of the alkalies and alkaline earths are
soluble both in water and in alcohol, those of the heavy metals are
mostly much more insoluble in water, so that addition of salts of such
metals as lead, copper, iron, or silver, causes precipitation of the corre-
sponding glycocholates. The lead salt is soluble in rectified spirit, from
which it is precipitated on the addition of water. The acid and its
salts possess a peculiar taste, sweetish at first, but afterwards intensely
bitter.

Tmiroclwlic acid (CjeH^gNSOy), also a monobasic acid, is crystallisable
with difficulty, forming fine deliquescent needles. It is very easily soluble
in water, and also possesses the power of carrying glycocholic acid into
solution when that acid is simultaneously present. It is exceedingly
soluble in alcohol, but insoluble in ether. In solution it possesses a
bitter-sweet taste, which is shared by its alkaline salts. The salts are
generally easily soluble in water, and a solution of an alkaline tauro-
cholate, unlike that of a glycocholate, is not precipitated by the usual
salts of the heavy metals, such as copper sulphate, silver nitrate, or
neutral lead acetate ; basic lead acetate does, however, precipitate it, and
the compound so formed is soluble in boiling alcohol.

Taurochohc acid is not nearly so stable a compound as glycocholic
acid, it decomposes on boiling in aqueous solution, or in evaporating to
dryness ; hence the dry pure acid has never been prepared or analysed,
and its formula has been deduced from analogy with glycocholic acid,
and from analyses of its more stable salts. Its solutions rotate the
plane of polarisation to the right, like glycochoHc acid. The specific
rotation of the alcohohc solution of the sodium salt is +24°'5. Potassium
taurocholate occurs in the bile of many fishes ; it possesses the peculiar
property of being completely thrown out of solution in water by the
addition of solution of caustic potash, and so may be prepared by adding
this reagent to an aqueous solution of an alkaline taurocholate.
Analyses of tliis salt by Strecker^ established its formula as
C26H44KNSO7, and analyses of the sodium salt gave a corresponding
result, from which it follows that the formula of taurochohc acid
itself is C20H45NSO,.

Hyoglycocliolic acid is an acid found in pig's bile,^ which yields on decora-
position glycocoll, like ordinary glycocholic acid, but an acid differing in
composition and behaviour from ordinary cholalic acid (Co4H^QOr,), and
called hyocholalic acid (C25H40O4). This acid differs from cholalic acid in not
being so easily crystallisable, and in having a difficultly soluble barium salt.
Severin Jolin '^ states that pig's bile contains, as principal bile salts, the sodium
salts of two different hyoglycocholic acids, each of which yields on decomposi-
tion glycocoll and a hyocholalic acid (a and /3). The two hyoglycocholic acids
are distinguished by the different solubilities of their sodium salts in neutral
salt solutions. The /3-salt is present in much greater quantity ; but the
distinguishing character of pig's bile, that it is precipitated by saturation with
various neutral salts, is not due to the fS- but to the a-hyoglycocholic acid.

^ Loc. cit.

^ Strecker and Grundelach, Ann. d. Chem., Leipzig, 1847-9, Bd. Ixii. S. 205 ; V>d. Ixx.
S. 179.

^ Ztschr. f. pJiysiol. Chem., Strassluirg, 1887-9, Bd. xi. S. 417; Bd. xii. S. 512;
Bd. xiii. S. 205.



THE BILE ACIDS. 377

The two hyocholalic acids show analogous differences to the two hyoglycocholic
acids. The formula of a-hyoglycocholic acid is C27H43NO5, that of ^-hyo-
glycocholic acid is CggH^gNOr,.

Tatorochenocliolic acid,^ the principal bile acid of goose bile, has the
formula CggH^gNSOg, has not been crystallised, and is soluble in water and
alcohol. From this acid Heintz and Wislicenus 2 prepared chenocholic acid
(C27H44O4) ; this is itself crystallisable with difficulty, but yields a barium salt,
which is insoluble in water and can easily be obtained in a crystalline form.

Pettenkofers test for bile acids.^ — When bile is gently warmed with
concentrated sulphuric acid and cane-sugar, a beautiful purple or
purplish-red colour develops, becoming deeper on standing. The colour
is due to an interaction between the bile salts, or cholalic acid, and a
substance called furfurol or furfuraldehyde developed by the action of
the strong sulphuric acid on the cane-sugar;^ hence the test may
be more satisfactorily carried out where only traces of bile salts are
suspected, by using a solution of furfurol (1 per 1000) instead of cane-
sugar.

To carry out the test in the ordinary manner, add to a drop or two of the
bile, or fluid suspected of containing bile acids, a drop of strong sulphuric
acid, taking care that any great rise in temperature does not occur 3 spread the
mixture out in a thin film in a porcelain capsule, and either add a drop of a
10 per cent, solution or a small crystal of cane-sugar ; if the violet colour does
not appear at once, warm very gently. To carry out the test with furfurol, one
drop of a solution of furfurol (1 per 1000) is added to 1 c.c. of an alcoholic
solution of bile salts, and 1 c.c. of concentrated sulphuric acid is added
cautiously to this, so as not to overheat. In this manner ^V^sV ^^ ^ milligramme
of cholalic acid may be detected.*^

The test with sugar may be easily spoiled by overheating or when
too much sugar is used, which favours carbonisation. The presence of
sulphurous acids or nitrous fumes in the sulphuric acid is also unfavour-
able to the reaction. Strong phosphoric acid may be used instead of
sulphuric acid.

Many other substances give a similar reaction. Pettenkofer himself
was aware that proteids gave a similar colour, though much less easily.
By subsequent observers ^ a large number of substances giving colour re-
actions with furfurol have been described ; amongst these many phenols
and aromatic bases are included, some of which are also found in
the urine, v. Udranszky ^ gives a list of over forty substances which
give colour reactions with furfurol, but none except a-naphthol show
the reaction with the same delicacy as the bile salts. That the
coloured substance so produced is not in all cases the same, is shown
by the fact that some possess no absorption spectrum, and that the
spectra of the others differ from one another. In this way the
spectrum of the colour given by the bile salts may be distinguished

1 Marsson, Arch. d. Fharm., Bd. Ivii. S. l.'^S.

2 Ann. cl. Phys. u. Chem., Leipzig, 1859, Bd. cviii. S. 547.

3 Ann. d. Chem., Leipzig, 1844, Bd. lii. S. 90.

4 Mylius, Ztschr. f. physiol. Chem., Strassburg, 1887, Bd. xi. S. 492.
s V. Udranszky, ibid., 1888, Bd. xii. S. 355.

^ Baeyer, Ber. d. deulsch. chem. Gesellsch., Berlin, 1872, Bd. v. S. 26 ; Stenhouse,
Ann. d, Chem., Leipzig, 1870, Bd. clvi. S. 197 ; Schiff, ihid., Bd. cci. S. 355.
'' Loc. cit. Drechsel, Journ. f. pralct. Chem., Leipzig, Bd. xxvii. S. 424.



378 CHEMISTR Y OF THE DIGESTIVE PROCESSES.

from the others by two bands, one between the solar lines D and E
near to E, the other at Y}

The bile salts ^Droduce great slowing of the heart's beat, which may be used
as a physiological test for them in confirmation of Pettenkofer's reaction. In
a curarised frog the heart is exposed, the pericardium removed, and the action
of the vagus paralysed by atropine; on now adding a drop of a solution of a
bile salt, the rhythm of the heart is greatly slowed.-

Cleavage products of the bile acids. — All the bile acids, under the
action of hydrating agents, split up into two components, of which one
is always either glycocoll or taurin, and the other a non-nitrogenous
monobasic acid which may be cholalic acid or one of several allied acids.

Glycocoll and taurin are nitrogenous bodies, belonging to that class
of substances called amido-acids, i.e. organic acids, in which one or more
hydrogen atoms are replaced by the group amidogen (NH2). Both these
amido-acids are probably formed by the breaking up of proteids, or
their allies the albuminoids.

The process of hydration can be carried out directly from bile, by heating
with hydrochloric acid, in a flask attached to a reversed condenser. Taurin
and hydrochlorate of glycocoll are formed, and the free cholalic acids, which
slowly lose M^ater and pass into the form of their anhydrides (the dyslysins,
p. 382) ; these being insoluble are precipitated. As soon as the reaction is
completed, as shown by the failure of Pettenkofer's test, the flask is allowed
to cool and the dyslysins filtered off. The filtrate, which contains the
amido-acids, is strongly concentrated, and, while still warm, decanted from the
sodium chloride which has crystallised out. It is next evaporated to com-
plete dryness and treated with absolute alcohol, which takes up the glycocoll
hydrochlorate and leaves the taurin behind. The residue is dissolved in as
small a quantity as possible of warm water, and filtered while warm ; to
this filtrate a little alcohol is added, and, on slowly cooling, crystals of taurin
are formed.

The alcohol is evaporated from the alcoholic extract containing the glycocoll
hydrochlorate, and water is added ; to the watery solution, hydrate of lead is
added, when insoluble lead chloride and a soluble lead compound of glycocoll
are formed. The latter is separated in solution by filtration ; into the solution
a stream of sulphuretted hydrogen is passed, the lead sulphide is filtered off,
and the filtrate is concentrated, until, on cooling, free glycocoll crystallises out.

The free cholalic acids ^ can be recovered from the dyslysins formed in
the first step of the above process. The dyslysins are removed from the
filter, and boiled with dilute alkali, when they take up water, and, combining
with some of the alkali, are converted into soluble alkaline cholalates. On
acidifying with hydrochloric acid, and evaporating to dryness, the cholalic
acids can be extracted with a small quantity of hot alcohol, from which they
crystallise on cooling, or on the addition of excess of ether.

Glycocoll, fjlycocine, or glycine, is amido-acetic acid (NHo.CHlCOOH).
Besides occurring combined with cholalic acid, as glycocholic acid in the
bile, it is found in the urine of certain animals and occasionally in man,
combined with benzoic acid, to form hippuric acid, and is formed as an
end hydration product from gelatine and similar substances.

1 Kosclilakoff and Bogomoloff, Ccniralhl. f. d. mcd. Wissensch., Berlin, 1868, BJ. vi.
S. 529. In this paper four bands are described. Bogomoloff, ibid., 1869, Bd. vii. S. 529 ;
Schenck, Jahresh. il. d. FoHschr. d. Thier-C'hem., Wiesbaden, 1872, Bd. ii. S. 232.

^ Mackay, Arch. f. exper. Path. u. Pharmakol., Leipzig, 1885, Bd. xix. S. 279.

^ The term "cholalic acids " is used to signify cholalic acid and its allies.



CLE A VAGE PRODUCTS OF THE BILE ACIDS. 379

It crystallises in colourless rliombohedra, or in four-sided prisms, which have
a sweet taste and dissolve easily in cold water (1 in 4-3) ; in alcohol and ether
they are insoluble. Glycocoll, like other amido-acids, can act chemically, either
as a base or an acid in forming compounds with acids and bases respectively.
As a type of these combinations with bases, the copper compound may be
taken. When freshly precipitated cupric hydrate is added to a warm con-
centrated solution of glycocoll, it dissolves to form a deep blue solution, which
is not reduced on boiling ; on cooling this solution, or on adding alcohol and
allowing to stand, fine dark blue needles crystallise out of the composition
(NH2.CH2.C02)2Cu,H20. Glycocoll has been obtained synthetically by the
action of ammonia on monochloracetic acid thus : —

Is^H, + CH2.CI— COOH = CH2.(NH,)— COOH + HCL

Taurine is amido-isethionic acid, also called amido-oxyethylsiilphonic
acid (N"H2.C2H4.S020H).i It occurs in the body, apart from the bile, only



Online LibraryE. A. (Edward Albert) Sharpey-SchäferText-book of physiology; (Volume v.1) → online text (page 53 of 147)