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

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in minute and inconstant traces ; it has been stated to occur in the lungs
and kidneys of oxen, in some of the organs of cold-blooded animals, and
in inconstant traces, probably due to the decomposition of taurocholates,
in the intestine. The presence of sulphur in its molecule shows it to be
formed from proteids in the body; but the intermediate steps in its
formation are unknown.

Taurine is very easily crystallised, and forms large colourless prismatic
prisms with a glassy giance,^ without any taste, and gritty between the teeth,
neutral in reaction and very stable, not being altered by a temperature of
240 °C. ; heated above this temperature they melt and decompose in so doing.
It is much less soluble in cold water than glycocoll, but still easily soluble
(1 in 15-5), and still more so in hot water; in alcohol and ether it is insoluble.
It is soluble in concentrated sulphuric and nitric acids without decomposition,
and the latter acid may even be boiled off, leaving it unaffected ; neither is it
affected by boiling with aqua regia. To alkalies also it is much more stable
than glycocoll; it is not affected by weak alkalies, and only by continued
boiling in strong alkaline solution is slowly broken up into ammonia, acetic,
and sulphurous acids ; so that it is one of the most stable of the organic
compounds found in the body. Taurine is also a much more neutral substance
in its chemical behaviour than glycocoll ; it does not combine at all with acids,
and its affinity for bases is very feeble. An amorphous mercury compound is
however obtained by boiling a solution of taurine with freshly-precipitated
mercuric oxide. ^

The constitution of taurine is shown by its synthesis from chlorethyl-
sulphonic acid by the action of ammonia.*

/C^H.Cl /C,H,NH.,

S0.< +.,NH3 = S0/ +NH,C1

This synthesis, as well as the fact that taurine is not saponified by
dilute alkahes, shows that taurine is not an ester but a sulphonic
derivative ; that is, that the sulphur atom is united directly to carbon,
and not indirectly by oxygen. Taurine may also be obtained by heating

1 The amido-oxyethyl radicle is directly uuited to sulphur in the molecule.
^ Gmelin, " Verdauung nach A^ersuchen," S. 60.

^ Lang, Jahresh. il. d. Fortschr. d. Thicr-Chem., Wiesbaden, 1876, Bd. vi. S. 74.
•^ Kolbe, Ann. d. Chem., Leipzig, 1862, Bd. exxii. S. 33.


the ammonium salt of oxyethylsulphonic acid to 230° C, when a
molecular rearrangement takes place, thus :

so/ =S0/ +H,0

^ONH, \0H

Cholalic acid is the usual partner with glycocoll or taurine in the
formation of the bile acids ; it is also found in the intestinal contents,
and sometimes, in cases of jaundice, in the urine. One method of
obtaining it has already been incidentally mentioned, but it is better
prepared by the following method : ^ —

Ox bile is boiled for about twenty-four hours with the fifth part of its
Yolume of 30 per cent, caustic soda solution, the water being replaced as it is
removed by evaporation. The solution is then saturated with carbon-dioxide
gas, and evaporated almost to dryness. The residue is extracted with 96 per
cent, alcohol, and the extract, diluted so that it does not contain above 20
l^er cent, of alcohol, is completely precipitated with a solution of barium chloride.
The precipitate, which consists of impure barium choleate, is filtered off, and
cholalic acid is precipitated from the filtrate by the addition of hydrochloric acid.
The acid slowly becomes crystalline on standing, when it is separated and puri-
fied by repeated recrystallisation from alcohol. Cholalic acid occurs in many
crystalline forms. ^ Anhydrous crystals forming flat- ended 4- to 6-sided prisms,
may be obtained by dissolving the amorphous form of the acid, produced by
drying one of the other crystalline forms, in ether and allowing the solution
to crystallise out.

From strong alcohol the acid crystallises, on the addition of a very little
water, in octohedra and tetrahedra, belonging to the orthorhombic system, and
containing two and a half molecules of water of crystallisation ; from dilute alcohol
it crystallises in fine shining flat needles or plates, containing only one molecule
of water of crystallisation.^ It also crystallises in large rhombic tetrahedra, or
octohedra containing one molecule of alcohol. Pure anhydrous cholalic acid
melts at 194° to 195° to a colourless liquid; and on heating above this temjDerature,
loses water and is converted into its anhydride or dyslysin ; on further heating,
it loses more water and yields a viscid yellow or, yellow-brown oil with a
green fluorescence ; this is another anhydride, with the composition C48HgQO.;;.

All forms of the acid are sparingly soluble in water and ether, and easily
soluble in alcohol. The solutions possess the bitter-sweet taste of bile. The
alkaline salts are crystalline and soluble in water, but precipitated by strong
solutions of alkalies or their carbonates. The barium salt is much more
soluble in cold water (1-30) than the corresponding salts of the allied acids
described below. Cholalic acid and its soluble salts turn the plane of polarisa-
tion to the right.'* Methyl and ethyl ethers of cholalic acid have been

The formula of cholalic acid was first established by Strecker^ as
C24H40O.-, and this formula, after some dissent,^ is now generally
accepted. AVith regard to its constitution, in spite of a vast amount of
labour on the subject, we are still only possessed of very fragmentary
and uncertain information. It is certainly a monobasic acid, and must

1 Mylius, Ztschr.f. pliysiol. Cheni., Strassbnrg, 1888, Bd. xii. S. 262.
- See Maly, Hermann's " Handbuch," Bil. v. (2), S. 136.

^ Schotten, Ztsclir. f. 'phydol. Chem., Strassburg, 1886-7, Bd. x. S. 175 ; xi. S. 268.
•* Hoppe-Seyler, Jonrn. f. pralct. Ghem., Leipzig, 1863, Bd. Ixxxix. S. 265; E. Vahlan,
Ztschr. f. 2}hysLol. Chem., Strassbnrg, 1896, Bd. xxi. S. 253.
^ Loc. cit.
® LatscliinofF, Ber. d. dcutsch. chem. GescUsch., Berlin, 1887, Bd. xx. S. 1968.


therefore contain one carboxyl group (COOH), and according to Myliiis ^
it also contains one secondary (CHOH) and two primary alcohol
groups (CH2OH).

The evidence for this is derived from its behaviour on cautious
oxidation. It first yields, when oxidised, monobasic dehydrocholalic acid
(C^4H3405),2 and on further oxidation tribasic bilic, or bilianic acid
(0,4113408).^ These changes may be expressed by supposing that, in
the formation of dehydrocholalic acid, the two primary alcohol groups
form aldehyde groups (H — C = 0), and the secondary group, a ketone
group (C = 0), and that in the further formation of bilianic acid the two
aldehyde groups pass into (acid or) carboxyl groups, so producing a
tribasic acid ; while besides, in the rest of the molecule, an additional
ketone group is formed, as shown by the following formula; : —

Cholalic acid, C,oH3i(CHOH)(CH.,OH),(COOH), on oxidation forms,
in place of one secondary alcohol group (CHOH) and two primary
alcohol groups (CHgOH), one ketone group (CO) and two aldehyde
groups (COH), thus yielding dehydrocholalic acid, C2oH3i(CO)(COH)2
(COOH), in which, on further oxidation, an additional ketone group is
formed, and the aldehyde groups change into carboxyl groups (COOH),
thus yielding the tribasic acid, bilianic (or bilic) acid, Ci9H3i(CO.)2(COOH)3.

Scarcely anything is known of the arrangement of the atoms in the
hydrocarbon part of the molecule. Mylius^ has obtained a reaction
between cholalic acid and iodine, in solution, with the formation of a
blue compound, which is crystallisable and becomes easily dissociated in
the same manner as iodide of starch. For example, in solution, it
becomes decolorised on heating.

This substance is probably an addition product of cholalic acid and
iodine, and so points out that the hydrocarbon radicle of the acid is not
fully saturated ; beyond this, however, we know nothing of its composition.

Desoxycliolalic acid is a reduction compound obtained by Mylius ^ of
the formula C24H40O4.

Clioleic acid '^ was first found in the preparation of cholalic acid from ox
bile, and separated from it by means of the more sparing solubility of its barium
salt. According to Lassar- Colin, it also occurs in human bile, and its formula
is C.,4H4|,04. Latschinoif,''' its discoverer, ascribed to it the formula C25H40O4.
From Lassar-Cohn's ^ formula it appears to be isomeric, or perhaps identical,
with desoxycholalic acid.

Fellic acid ^ (C23H4QO4) is an acid which has been obtained from human
bile ; it is crystalline, insoluble in water, and forms insoluble barium and
magnesium salts.

The acids formed by the cleavage of the peculiar bile acids found in the bile
of the pig and goose have been already mentioned in treating of these acids.

^ Bar. d. deutsch. chem. Gesellscli., Berlin, 1886, Bd. xix. S. 369, 2000.

^ Hammarsten, ibid., 1881, Bd. xiv. S. 71 {Dehydrocholsailre) ; Lassar-Colm, ibid., 1892,
Bd. XXV. S. 805 ; Ztschr. f. physiol. Chem., Strassburg, 1892, Bd. xvi. S. 488.

^ Cleve, Bull. Soc. cJiivi., Paris, tome xxxv.

^ Ztschr. f. pliysiol. Chem., Strassburg, 1887, Bd. xi. S. 306 ; Bcr. d. deutsch. chem.
Gesellsch., Berlin, 1887, Bd. xx. S. S83.

^ Loc. cit.

^ Choleinsdure of Latschinoff, Ber. d. deutsch. chem. Gesellsch., Berlin, 1885, Bd. xviii.
S. 3039.

'^ Loc. cit.

^ Ber. d. deutsch. chem. Gesellsch., Berlin, 1894, Bd. xxvii. S. 1339.

^ Fellinsdure oi Schotten, Ztschr. f.physiol. Chem.. Strassburg, 1887, Bd. xi. S. 268. See
also Lassar-Cohn, ibid., 1894, Bd. xix. S. 563.


The acids formed as cleavage products from hiiman bile are cholalic,
choleic, and fellic acids.

Cholalic acid and its allies, on boiling with acids, on heating in the
dry state, or by putrefaction, lose water, and become converted into
anhydrides, or, as they are called, dyslysins. The dyslysin corresponding
to ciiolahc acid has the formula C04H06O3 ; it is found in faeces ; is a white
amorphous substance, msoluble in water and alcohol, soluble in ether
and melting at 140° C. Another compound, choloidinic acid, is formed,
as an intermediate stage, of the formula C24H38O4. On boiling with
alkalies, dvslysin takes up water, and is reconverted into cholahc acid.

The bile pigraents and their derivatives. — The variations in the
colom' of bile early attracted attention, and Gmelin,^ in 1826, first
obtained proof of a relationship between these colours, and described
the test which stiU bears his name. He was aware that the play of
colours was due to a process of oxidation, and made an experiment to
illustrate this by acidifying bile with hydrochloric acid, and enclosing
it in a tube from which the air was shut off by a mercury trap. Under
these ckcumstances no change in colour took place; but on exposing
the acidified bile to the air, a green colom- slowly developed. He also
accm-ately described the play of colours obtained on oxidising with
nitric acid.

Berzelius ^ precipitated biliverdin from ox bile with barium chloride,
purified it to some extent, and described its properties, but he feU into
the error of supposing that it was identical with chlorox^hyU-^

Heintz,* preventing oxidation by exclusion of air, extracted from
gallstones a brown amorphous pigment, which he named bilipham.
He analysed it, and converted it by dissolving in sodium carbonate, and
leading oxygen through the solution into a green pigment, bihverdin.
His biliphain corresponded to the bilirubin of the present day, and his
experiment shows well the connection between the two pigments.

Valentmer,^ in 1859, was the first to obtain bilirubin in a crystalhne
form, by dissohdng in chloroform, from which, on evaporation of the
solvent, it crystallises in microscopic crystals. From this discovery
onwards, research on the bile pigments took a more exact form, as
methods for the isolation of the pigments were discovered and perfected.^

Although a considerable numloer of more or less well-characterised
bile pigments have been described, only two are found under normal
conditions in the bile, these are bilirubin and JDiliverdin ; the others are
obtained by artificial means from these, are found under pathological
conditions only in the body, or are formed after death. The colour of
the bile is a compound of the colour of these two pigments, and varies
with the varying ratio of their amounts through all shades between

^ Tiedemann and Gmelin, "Yerdauung nach Versuclaen, " 1826.

- "Chemie," S. 281. '

^ The spectra of phylloporphyrin and hsematoporplij^rhi and their derivatives ai'e almost
identical, and in other respects the substances closely resemble each other, so that there is
undoubtedly a relationship between them (Schunck and Marchlawski, Proc. Roy. Soc.
London, Jan. 1896). Kow, jjhylloporphyriu is a derivative of chlorophyll, and hremato-
porphjTin is isomeric with bilirubin, so that there may be some remote connection between
biliverdin and chlorophyll.

^ Jahrcsb. ii. d. Fortschr. d. ges. Med., Erlangen, 1851, Bd. ii. S. 59; Ann. d. Phys.
u. Chem., Leipzig, 1851, Bd. Ixxxiv. S. 106.

5 Jahresb. ii. d. Fortschr. d. ges. Med., Erlangen, 1859, Bd. ii. S. 87.

« Briicke, Untersuch. z. JVaturl. d. Menscli. u. d. TMere, Bd. vi. S. 173 ; Stadeler,
Vrtljschr. d. natnrf. Gesellsch. in Zurich, 1863, Bd. viii. S. 1 ; Ann. d. Chem., Leipzig
1864. Bd. cxxxii. S. 323.


reddish-brown and grass-green. To the variation in relative amount of
these two pigments is also due the difference in colour between fresh
and stale bile. When bile stands in the gall jjladder, its pio-ments
become reduced, the biliverdin is converted into bilirubin, and the
colour becomes yellow or brown. Fresh human bile has also a green
colour, but that observed in the post-mortem or dissecting-room is
always brown, because of this process of reduction. These two normally
occurring bile pigments are related to each other in a manner analogous
to hteinoglobin and oxyhaemoglobin ; bilirubin (Ci(;Hig]Sr203) on oxidation
passes into biliverdin (CjgHigN.^Oi).

Haycraf t and Scofield ^ observed in the gall bladder itself reduction
going on, as shown by the fact that, while the bile in the middle of the
gall bladder was green, the thicker bile mixed with mucus near the
bladder wall was orange-brown, and the mucous membrane itself of a
brown colour. To this slow reduction Haycraft and Scofield ascribe
also the presence of bilirubin and not biliverdin in the gallstones of
oxen, although the latter is the chief pigment found in ox bile. Putre-
faction readily brings about the same reduction in the bile pigments.
Bile with the bilirubin tint predominant does not turn green from,
oxidation of this pigment to biliverdin, when it is exposed to the air,
unless it be made strongly alkaline with caustic alkali. In this increased
readiness to take up oxygen in alkaline solution, bihrubin resembles a
large number of other organic substances, such as pyrogallol and pyro-
catechin. Haycraft and Scofield were also able to induce these chano-es
by the action of nascent oxygen and of ozone. Working with a battery of
four or five Grove cells, and leading from platinum electrodes into brown-
coloured bile (in a beaker or on filter paper), they found that the oxyo-en
developed at the anode caused in a few minutes a change in colour of
the bile, through green and blue into violet, followed by bleaching. On
reversing the poles, so that reduction instead of oxidation took place at
this spot, an inverse change in colour back to brown was observed.

Bilirubin and biliverdin have chemically the character of weak acids,
as is shown by the ease with which they unite with bases to form salt-
like bodies. Such compounds with alkalies are soluble in water, but
with alkaline earths are insoluble; as, for example, the compound of
bilirubin with calcium, which makes up the bulk of red gall stones, and
forms a convenient source for the preparation of the pigment. Neither
pigment has a spectrum showing absorption bands, but in each there is
continuous absorption at the blue end of the spectrum.

Bilirvhin has borne in the history of the bile pigments many names,
such as cholepyrrhin, biliphain, cholephain, and bilifulvin; but, fortunately,
all these names have now disappeared, and the properties of the sub-
stances described under them by different observers, so far as they have
been substantiated, have been aggregated under one name and to one
substance, bilirubin, the red colouring matter of bile. Bilirubin is a con-
stant constituent of bile, and is found besides as a calcium compound in
red gall stones. It is also present in traces in the serum of some animals.
Hammarsten^ found it in the serum of the horse. By precipitatmo-
the serum globuhn with acetic acid, the pigment is throwm down with
the globulin, and on drying the precipitate and extracting with chloro-
form, bilirubin is dissolved out. It seems, however, to be absent in

'^ Ztsclir. f. physiol. Chem., Strassburg, 1889, Bd. xiv. S. 173.

^ Jahresb. u. d. Fortsclir. d. Thier-Chem., Wiesbaden, 1878, Bd. viii. S. 129.


human serum and that of the ox. Most important, from the point of
view of the origin of the bile pigments, is the discovery that the
microscopic crystals often found in old blood clots and extravasations,
and described by Virchow ^ as hsematoidin, are usually bilirubin ; this
shows that the bile pigments are probably products of disintegration of
haemoglobin. Here it is needful to guard against mistaking lutein for
bihrubin. The two substances may be distinguished by their solubilities.
Both are soluble in chloroform, but bihrubin is thrown out of solution
on the addition of an alkah (from the formation of a compound with the
alkali insoluble in chloroform), while lutein is not so precipitated.
Bilirubin is also found, in cases of jaundice, in the urine and in the

Bilirubin is best prepared from the gallstones of the ox, which are very
common and easily procurable. The gallstones are washed, dried, powdered,
and then extracted in turn with ether, boiling alcohol, and boiKng water, to
remove cholesterin (which is, however, rarely present in appreciable quantity
in ox gallstones) and bile acids. The residue is treated with dilute hydro-
chloric acid, to set free the bilirubin from its calcium combination, washed
with water and alcohol, and finally extracted with boihng chloroform, in which
the bihrubin dissolves.

The chloroform is distilled from the extract, and the impure bilirubin is
freed from an accompanying substance, bilifuscin, by digesting with absolute
alcohol, in which this substance dissolves, and is then redissolved in chloro-
form. It is purified further by throwing out of concentrated solution in
chloroform, by the addition of absolute alcohol, redissolving and reprecipitating
repeatedly. It is lastly dissolved in as little as possible of boiling chloroform,
from which it crystallises on cooling.

In amorphous condition, as when precipitated by alcohol and dried,
bilirubin is an orange-coloured powder ; when crystalline, it is of a dark red
or reddish-brown colour, resembling chromic acid. The crystals are rhombic
plates with rounded-off angles ; they are more easily soluble in chloroform
than in any other solvent ; somewhat soluble in carbon bisulphide and amyl
alcohol ; nearly insoluble in ether, alcohol, turpentine, benzol, and glacial acetic
acid. Bilirubin is soluble easily in alkalies and their carbonates, combining
with them to form salts. Calcium chloride, added to these solutions, pre-
cipitates the calcium compound (CjgHj^N20o).2Ca as a rust-coloured precipitate.
Treated with sodium amalgam, bilirubin yields hydrobilirubin ; on oxidation
it passes into biliverdin and more highly oxygenated compounds. Several
formuljB have been proposed for bilirubin ; - the most generally accepted is
that of Maly (C^gH^gNoOg). Bilirubin is oxidised in alkaline solution in the
air to biliverdin in the same manner as bile, which owes this reaction to the
bilirubin it contains.

Ehrlich's test.^ — Ehrlich describes a colour test for bilirubin, which is not
given by biliverdin. To a solution of bilirubin in chloroform an equal volume
of a watery solution of diazobenzolsulphonic acid is added, and just enough
alcohol to cause the two fluids to mix, when the fluid turns a beautiful red
colour ; on adding, drop by drop, concentrated hydrochloric acid, the colour of

^ Virchow' s ArcMv, 1847, Bd. i. S. 379, 407. See also Robin, Compt. rend. Acad. d. sc,
Paris, 1855, tome xli. p. 506 ; Jaff(5, Firchovis Archiv, 1862, Bd. xxiii. S. 192 ; E. Salkowski,
Hoppe-Seyhr's Med.-chcm. Untersuch. , Berlin, 1868, S. 436.

^ Stadeler, Ann. d. CJiem., Leipzig, 1864, Bd. cxxxii. S. 323 ; Thudiclmni, Journ. f.
praU. Chem., Leipzig, 1868, Bd. civ.'S. 401 ; Maly, ibid., Bd. civ. S. 28 ; Maly, Ann. d.
Chem., Leipzig, 1876, 13d. clxxxi. S. 106 ; Nencki u. Sieber, Ber. d. deutscli. chem. Gesellsch.,
Berlin, 1884, Bd. xvii. S. 2275.

^ Centralhl. f. Iclin. Med., Bonn, 1883, Bd. iv. S. 722; Krukeuberg, " Chenm. Unter-
such.," 1886, S. 77.


the solution changes through violet into blue ; if a layer of potassium hydrate
solution is now introduced beneath the blue solution, there develops an
(alkaline) bluish-green zone underneath, separated from the blue solution above
by a red band where the reaction is neutral.

Biliverdin is present in all green-coloured biles, and may be obtained from
them, by adding a solution of barium chloride, as a dark green precipitate,
which may be washed with water and alcohol, and decomposed by dilute hydro-
chloric acid, when the biliverdin remains as a flocky precipitate ; this is freed
from fats by washing with ether, and is then dissolved in alcohol ; on evaporat-
ing the alcohol, the biliverdin is left behind as a dark green scale. Biliverdin
can best be prepared pure from an alkaline solution of bilirubin. This is
allowed to oxidise by exposing to the air in a shallow dish, until it turns a
brownish-green colour ; the solution is then precipitated with hydrochloric
acid, which sets free insoluble biliverdin from the soluble compound with the
alkali ; the precipitate is washed with water till free from hydrochloric acid,
dissolved in alcohol, and reprecipitated by the addition of water. This
precipitate is washed with chloroform to remove traces of bilirubin, and pure
biliverdin remains behind, being insoluble in chloroform. It forms a very
dark green amorphous powder, insoluble in water, ether, chloroform, carbon
bisulphide, or benzol ; but soluble with a fine green colour in alcohol, glacial
acetic acid, or concentrated sulphuric acid. According to MacMunn,^ there is
a green pigment in ox bile which differs from that prepared as above, in being
soluble in chloroform. Biliverdin does not easily crystallise ; it is said to be
occasionally obtainable, by evaporating a solution in glacial acetic acid, in
rhombic plates with rounded angles. "With alkalies, biliverdin forms soluble
compounds, giving brownish-green solutions, from which biliverdin falls as a
flocky precipitate on the addition of acids. Calcium, barium, and lead salts
form insoluble compounds with biliverdin ; these are thrown down as dark
green precipitates on addition of solutions of the corresponding salts to an
alkaline solution of biliverdin. By nascent hydrogen, biliverdin is converted
through bilirubin into hydrobilirubin. Different formulae are given by different
authors for biliverdin. Stadeler ^ calculated it as C^gHgolSroOg, from analyses
by Heintz. Heintz himself ^ gives CjgH^glSr^Oj. This formula assumes that
in passing from bilirubin to biliverdin, the molecule takes up water as well as
oxygen (Ci,.,H^gN"203 -1- H.3O -f = C;i5H2o]Sr205), but the accuracy of this is
denied by Maly,^ both from analysis and the amount of increase in weight
observed in passing from bilirubin into biliverdin. He gives the formula of
biliverdin as C^QHjg]Sr204, and this result is confirmed by Thudichum,^ except
that the latter halves the formula, giving CgHgN"02.

Gmelins test for bile jngmcnts. — This very distinctive test for the bile
pigments has ah^eady been mentioned. It depends upon the remarkable
changes in colour accompanying the oxidation of bilirubin. In such
oxidation the other normal bile pigment, bihverdin, is first produced ;
and this in turn, by further oxidation, is converted into a blue pigment,

Online LibraryE. A. (Edward Albert) Sharpey-SchäferText-book of physiology; (Volume v.1) → online text (page 54 of 147)