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bilicyanin; after this follows, according to some, a purple pigment
(bihpurpurin) before the final stage of oxidation to a yellow compound,
choletelin. The production in series of these artificial products of
oxidation is what constitutes Gmelin's test.

If either a solution of bilirubin or some diluted bile be carefully

^ Joivrn. Physiol., Cambridge and London, 1885, vol. vi. p. 25.

" Ann. d. Chem., Leipzig, 1864, Bd. cxxxii. S. 323.

" Jahresb. u. d. Fortsclir. d. ges. Med., Erlangen, 1851, Bd. ii. S. 59 ; Ann. d. PJiys.
10. Chem., Leipzig, 1851, Bd. Ixxxiv. S. 106.

'^Jahresb. il. d. Fortsclir. d. Thier-Chem., Wiesbaden, 1874, Bd. iv. S. 302 ; and Her-
mann's "Handbuch," Bd. v. (2), S. 159.

^ Jmcrn. f. prakt. Chem., Leipzig, 1868, Bd. civ. S. 220.

VOL. I. — 25



386 CHEAilSTR V OF THE DIGESTIVE PROCESSES.

poured on the surface of fuming nitric acid in a test tube, so as not
to mix the two liquids, a series of coloured zones appears in the
lower part of the column of bile above the acid ; next to the acid is the
most oxidized product (choletelin), represented by a yellow-red zone ;
above this is a purer red, passmg into a purple, which is replaced by a
blue zone (bilicyanin) ; and, lastly, there is a very broad green zone,
corresponding to the least oxidized product (biliverdin). The test may
also be made by spreachng out the bile in a thin film over the mside
of a j)orcelain capsule and placing a drop of fuming nitric acid in the
centre of this film, when a series of colours develop in the above order
around the drop ; or perhaps, most conveniently of all, according to
Eosenbach's modification, by moistening a piece of filter paper in the
suspected fluid, and placing a drop of fuming nitric acid in the
centre.

Ruppert's ^fesiJ.— Huppert's test consists in precipitating calcium
bilirubinate, by the addition of milk of lime, or calcium chloride and
ammonia, to a solution of an alkahne bilirubinate (or alkahne bile) ;
after washing with water, the precipitate is boiled for some minutes
with alcohol acidified with sulphuric acid, when, in presence of bile
pigments, the solution develops an emerald-green or blue-green colour.

Bilicyanin is the name given to the substance present at that stage of
oxidation of bilirubin by artificial means, such as fuming nitric acid, when
the solution has a blue colour. The stage is a very transient one, and, though
many have worked at the subject, no one has yet succeeded in isolating the
substance to which the blue colour is due. It is probably an unstable
oxidation product, intermediate between biliverdin and choletelin. A blue
solution, which keeps for some hours, may be obtained by adding to a solution
of bilirubin in chloroform a little nitric acid, and shaking till a violet tint
first appears. Rectified spirit is then quickly added ; this very much slows the
completion of the oxidation, so that the blue colour is preserved for some
time. If an ammoniacal solution of bilirubin be mixed with strong fuming
nitric acid, a little at a time, and excess of acid removed each time by addition
of ammonia, a dark flocky precipitate is obtained, from Avhich biliverdin can
be removed by alcohol, leaving behind a deep dark blue powder.^ Heynsius
and Campbell ^ have found that certaur gall stones in man, after extraction
with alcohol and ether, yield to dilute acids a violet-brown pigment, which
they identified as bilicyanin spectroscopically.

Jaffe ^ first observed that the blue stage of the oxidation process
gave an absorption spectrum ; in strong solution, it shows a wide band
beginning to the red side of D, and ending between D and E ; on dilution
this band resolves itself into two dimmer bands (a and |S). As oxida-
tion proceeds, a third band (7) appears between & and F, whilst the two
first mentioned gradually become fainter and disappear. This third
band does not belong to the blue stage (bilicyanin), but to the substance
formed in the final stage of the oxidation (clioletelin). The violet
colour obtained before the final permanent reddish brown is probably
due to an admixture of the latter colour with blue.

Bilifuscin is a substance separated in the preparation of bilirubin from
gall stones ; very little is known of its properties or chemical relationships.

^ Jaffe, Centralbl. f. d. med. Wissensch., Berlin, 1868, Bd. vi., S. 241; Journ. f.
2)rakl. C'hem., Leipzig, 1868, Br], civ. S. 401.

^ Arch. f. d. ijes. Physiol., Bonn, 1871, Bd. iv. S. 537. ^ Loc. cit.



BILE PIGMENTS AND THEIR DERIVATIVES. 387

In the presence of bilirubin it is soluble in chloroform, although diffi-
cultly soluble in this solvent alone ; hence, after treatment with chloroform in
preparing bilirubin, both substances come into solution. When the chloroform
solution is concentrated, and excess of alcohol added, the bilirubin is pre-
cipitated, while the bilifuscin remains soluble, and is found in the alcoholic
filtrate along with some cholesterin and higher fatty acids. After removal of
the alcohol by evaporation, the residue is treated with ether, which dissolves
out these impurities, and chloroform, which removes any traces of bilirubin
left behind. The almost black, dark brown residue so obtained, was termed
bilifuscin by Stadeler ; ^ who made incomplete analyses of it, from which he
deduced the formula CigHgoNaO^ (?). When quite pure, bilifuscin does not
give Gmelin's reaction ; - it is found in very old post-mortem bile ° as well as
in gallstones, but not in fresh bile. Bilifuscin has only been obtained in an
amorphous form ; it is soluble in alcohol and in alkalies ; almost insoluble in
water, ether, and chloroform ; its relationship to bilirubin is unknown. The
biliprasin of Stadeler'* is probably only a mixture of bilifuscin and biliverdin.
Bililiumin is a name used by the same observer to designate a black mass
taken up by strong solution of ammonia, from the residue of gallstones
which have been thoroughly exhausted with chloroform, alcohol, and ether ;
it does not give Gmelin's reaction.

Hyclrobilirubin (0321140^^07), a reduction product of bilirubin, is an
important substance, from the connection it makes between the bile
pigments, those of the urine and the products of disintegration of
haemoglobin.

Maly ^ first obtained it by the action of nascent hydrogen (from sodium
amalgam) on an alkaline solution of bilirubin ; biliverdin similarly treated
also yields it, being first converted into bilirubin. At the end of the reaction
the light brown coloured fluid is decanted from the mercury, and acidified
Avith hydrochloric acid. On the addition of the acid the solution becomes
much darker in colour, and abundant dark brown flocks of hydrobilirubin
separate out; these are separated from the solution, dissolved in ammonia,
reprecipitated with hydrochloric acid, and washed with water. After so
washing away all the salts the pigment becomes less soluble in water. After
drying it forms a dark reddish-brown powder, easily soluble in alcohol, or a
mixture of alcohol and ether ; not so soluble in ether alone. These solutions
have, when concentrated a reddish brown, when dilute a rose colour.
Chloroform dissolves it to form a yellowish-red solution. In alkalies it
dissolves to a pale yellow solution, becoming red on the addition of an acid.
Maly ascribes the yellow colour to a compound with the alkali, the red to
the free substance.

Hydrobilirubin in solution has an absorption spectrum, showing a
dark band between h and F. On addition of ammonia this band fades
out, but reappears a little to the left on the addition of a trace of zinc
chloride to the solution. This solution containing zinc chloride and
ammonia has a rose colour and a green fluorescence. Hydrobilirubin
once formed does not readily give Gmelin's test ; that is to say, it is not
easily oxidisable again to bilirubin or biliverdin.

Maly recognised his new substance as identical with a urinary

^ Vrtljschr. d, naturf. Gesellsch. in Zurich, 1863, Bd. viii.

2 Brllcke, Untersuch. z. Naturl. d. Menscli. u. d. TMere, 1860, Bd. vi. S. 173.

" Simony, Jahresl. it. d. Fortschr. d. Thier-Chem., Wiesbaden, 1876, Bd. vi. S. 75.

* Loc. cit.

^ Jahresh. il. d. Fortschr. d Thier-Chcm., Wiesbaden, 1872, Bd. ii. S. 232.



388 CHEMISTR Y OF THE DIGESTIVE PROCESSES,

pigment, already described under the name of urobilin, and discovered
by Jaffe ^ under pathological conditions in the urine. Immediately
before Maly's discovery, Hoppe-Seyler ^ had described a brownish-red
substance, wMch he obtained by the action of zinc and hydrochloric
acid {i.e. nascent hydrogen), on hgematm ; this, he afterwards stated to
be impure hydrobilirubin.^ When one considers that bilirubin is poured
into the intestine with the bile, and that it is here subjected to reducing
influences, as is shown by the frequent presence of hydrogen in the
intestinal gases, it is natural to suppose that a considerable conversion
of bihrubin into hydrobihrubin goes on in the intestine. The pigments
of the faeces, which must arise mainly from the bile pigments, do not
give Gmehn's reaction ; extraction of the fseces with dilute spirit,
evaporating and extracting the residue with strong spiiit, yields a
solution which shows the spectrum of hydrobilnul^in.'^ This pigment
of the fasces had been already described as stercobilin by Vanlair and
Masius.^ Jaffe^ considered it as identical wdth his urobihn. Maly
gives the above theory of its formation in the intestine from bilirubin,
and looks upon all three, as well as Hoppe-Seyler's compound from
haematin, as one substance, namely, hydrobilirubin.'^

It is generally accepted that these substances are closely related, if
not identical, and their relationsliip is of the utmost importance in con-
necting the pigments of the bile with the waste products of haemoglobin.^

Cholctelin. — Besides this important reduction derivative of bilirubin,
we also owe to Maly^ the discovery of choletelin, the final substance
obtained in its oxidation by nitric acid.

At the end of the reaction a yellow colour is obtained, not widely
different from that of the bilirubin from which the reaction started;
when this condition is reached, all the intermediate products have been
converted by oxidation into one substance.

Choletelin is best prepared, according to Maly,^" by leading a stream of
nitrous fumes (prepared by acting on arsenious acid Avith nitric acid) through
bilirubin suspended in alcohol. The fluid passes through the colours of
Gmelin's reaction, and finally a clear, pale, yellowish-red solution is left ; this
is thrown into water, when choletelin sejDarates out ia rust-coloured flocks,
which form, when dried, a brown powder. Choletelin is amorphous and
probably represented by the formula Cii3HisN20g ; it is soluble in alcohol,
ether, chloroform, and acetic acid. It is also soluble in alkalies, and
precipitated from such solution by acids. In acid solution it shows a dim
absorption band lying between h and F, and corresponding to the band y
observed by Jafi'e in solutions of bilicyanin. In neutral alcoholic solution this
hand disappears.

1 Virchovj's Arcliiv, 1869, Bd. xlvii. S. 405-418.

" Jahresb. ii. d. Fortschr. d. TMer-Chem., Wiesbaden, 1871, Bd. i. S. 80 ; Mcd.-chem.
Untersvch., Berlin, 1871, S. 536.

^ Jjer. d. deuisch. chem. Gcsellsch., Berlin, 1874, Bd. vii. S. 1065.

4 Maly, Hermann's "Handbueh," Bd. v. (2), S. 162.

^ Jahresb. ii. d. Fortschr. d. Thier-Chcm., Wiesbaden, 1871, Bd. i. S. 229.

« Arch./, d. ges. FhysioL, Bonn, 1871, Bd. iv. S. 537.

■^ Hei-maiin's "Handbueh," Bd. v. (2), S. 162.

** See MacMunn, Journ. Physiol., Cambridge and London, 1889, vol. x. p. 71 ; Eichholz,
Journ. Physiol., Cambridge and London, 1893, vol. xiv. p. 326 ; Garrod and Hopkins,
Journ. Physiol., Cambridge and London, 1896, vol. xx. p. 113 ; Garvoch, ihid., 1897, vol.
xxi. p. 190.

'^ Sitzungsb. d. k. Akad. d. Wissensch., Wien, 1868, Bd. Ivii. Abth. 2, S. 107; 1869,
Bd. lix. Abth. 2, S. 602.

" Hermann's "Handbueh '' Bd. v. (2), S. 165.



BILE PIGMENTS AND THEIR DERIVATIVES. 389

The halogens react energetically with bilirubin, forming substitution
products. When bromine acts on bilirubin, a series of changes in colour
take place, exactly counterfeiting those observed in Gmelin's reaction.
The reactions are, however, quite different ; and the process is not, as one
might naturally have expected, an oxidation, but a substitution of
bromine for hydrogen 1 {2{]^^^^.f)^-V?j^v^^Q^^^^x^,(d^-VZW^xY

The reaction is best shown by adding a dikite solution of bromine in
chloroform, cautiously, to a solution of bilirubin, also in chloroform. The solu-
tion changes through green, blue, and red into yellow, as the bromine is added,
and may be stopped and examined at any stage. If chloroform free from
alcohol be used, a tribromo derivative separates out of solution. Decanted
from chloroform, dissolved in alcohol, and reprecipitated by adding water, this
compound is obtained as a dark blue powder, soluble in alcohol, in ether, or
in chloroform containing alcohol ; but insoluble in pure chloroform, or in
water. Alkalies split it up, yielding biliverdin.

Nothing is known of the chemical constitution of the bile pigments,
and very little of the intermediate stages in their production from
hsemoglobin. Their connection with haemoglobin rests on — (1) The
identity of hsematoidin produced in old blood clots with bilirubin. (2)
The identity of Hoppe-Seyler's reduction product obtained from hsematin,
and thus indirectly from haemoglobin, with Maly's hydrobilirubin.

(3) The absence of bile pigments in such animals as have no hsemoglobin.^

(4) The fact that anything causing increased destruction of red blood
corpuscles, or the intravenous injection of hsemoglobin, causes an increased
secretion of bile pigments.* (5) Hasmatoporphyrin is isomeric with
bilirubin, shows with nitric acid colour changes somewhat resembling
Gmelin's reaction, and yields on reduction with nascent hydrogen a
substance closely resembling and probably isomeric with hydrobilirubin.
According to Nencki and Sieber,^ bilirubin is formed in the liver by the
hsemoglobin first splitting up into hsematin and proteid. The hsematin
thus formed then takes up water, loses its iron, which is retained in
combination in the liver, and so forms bilirubin, thus —

Cs^Hg^NAFe-h 2H2O— Fe = C32H36N,06; or 2(CicHi3¥203)

(hsematin) (bilirubin)

Direct experiments on the formation of bile pigments from hsemo-
globin apart from the liver have been carried out by Latschenberger.^
If the corpuscles and serum of horse blood be separately injected sub-
cutaneously at different parts in the horse, and after the lapse of about
twelve days the animal be killed, and the parts where the injections have
been made are examined, it is found that while, at the part where the

1 Thudichum, Journ. Chevi. Soc, London, 1875, vol. xxviii. p. 389 ; Maly, Sitzungsh. d.
k. Akad. d. TVissensch., Wien, Bd. Ixxii. Abth. 2.

2 Maly, Hermann's "Handbucli," Bd. v. (2), S. 167.

^ Hoppe-Seyler, Arch. f. d. cjes. Physiol., Bonn, 1877, Bd. xiv. S. 399. See, however,
Krukenberg, Centralhl. f. d. med. Wisscnsch., Berlin, 1883, No. 44, S. 785.

■^ Freriehs, Arch. f. Anat. u. Physiol., Leipzig, 1856, S. 59 ; W. Klthne, Virchoiv s Archiv,
1858, Bd. xiv. S. 310 ; Nothnagel, Berl. klin. Wchnschr., 1866, vol. vi. S. 31 ; Tarchanoff,
Arch. f. d. ges. Physiol., Bonn, 1874, Bd. ix. S. 329 ; Minkowski and Bassorin, Arch. f.
expcr. Path. u. Pharmakol., Leipzig, 1887, Bd. xxiii. S. 145.

5 Ber. d. deutsch. chein. Gesellsch., Berlin, 1884, Bd. xvii. S. 2275 ; Monatsh. f. Chem.,
Wien, 1888, Bd. ix. S. 115 ; Arch. f. cxper. Path. u. Pharmakol., Leipzig, 1888, Bd. xxiv.
S. 430.

'^Monatsh. f. Chem., Wien, 1888, Bd. ix. S. 52; Sitzungsh. d. k. Akad. d. Wissensch. ,
Wien, 1888, Bd. xcvii. Abth. 2b, S. 15.



390 CHEMISTR Y OF THE DIGESTIVE PROCESSES.

serum has been injected the tissues present a normal appearance and
contain no bile pigments, on the other hand, round the spot where the
corpuscles have been injected, the tissues contain, besides fluid blood, a
substance in flakes, varjang in colour from dark orange to bright yellow,
composed of small spherical masses about a quarter of the size of red
corpuscles, wliich give Gmelin's reaction very readily. The same result
may be obtained on injecting crystallised hsemoglobin, suspended in
water; here granular masses of a greenish-yelloiv colour are obtained,
which also give Gmehn's reaction.

Spectra of bile. — A considerable amount of continuous absorption at
both ends of the spectrum is found on examining the bile of any animal,
but in some animals the bile also shows well-marked absorption bands. ^

Cliololicematin. — The most characteristic of these band-spectra is that
exhibited by ox or sheep bile which has stood for some time in contact with
air. This spectrum, according to MacMunn,^ " presents in a deep layer three
bands, in a thinner one four bands, and in a still thinner a fifth band at F is
visible." The spectrum is well seen in an alcoholic solution of evaporated ox
bile. Of the four well-marked bands, two lie close to the D line, on either side
of it ; a third lies in the red, immediately to the right of the C line ; and the
fourth covers the E and b lines. ISI'o pure material has yet been isolated,
so that it is not even known whether the spectrum is due to one or several
substances. MacMunn^ has obtained an amorphoris residue of a dark sap-
green colour, containing abundantly material which gives the spectrum, by
treating ox bile with absolute alcohol and acetic acid, alternately dissolving in
chloroform and ether, and washing the chloroform solution with water. This
material has been named cholohsematin by MacMunn, from its occurrence in
bile and its supposed origin from hsematin.

The spectrum is not exhibited by fresh ox or sheep bile,* but is first
developed on standing in contact with air, probably from a chromogen present
in the fresh bile. The bands near D first appear, to be followed much later by
the other two ; the appearance of the spectrum is not a result of putrefaction.^

Mac^Munn^ obtained a spectrum closely resembling that of hsematoporphyrin
by the action of sodium amalgam on cholohsematin, prepared as above indicated,
from which he argues that the latter is a derivative of hsematin.

The fresh bile of the mouse shows a well-marked band at F, corresponding
to the urobilin band ; and more or less distinct bands in the same position in
the bile of other animals indicate, according to MacMunn, traces of urobilin
in these fluids. Characteristic absorption-band spectra are also found in the
bile of the guinea-pig, pig, rabbit, and crow. Human bile shows no bands, but
an alcoholic extract exhibits a well-marked band at D ; these, as well as the
spectra of Gmelin's and Pettenkofer's reactions, are shown in Plate III. at
the end of this volume.

Other constituents of bile. — Besides the bile salts and bile pig-
ments, which are normally found only in the bile, other constituents are
present which are also found in other parts of the body; these are
cholesterin, fats, soaps, and lecithin, besides minute traces of urea and
of the diastatic ferment already mentioned.

1 Bogomoloff, Centralhl. f. d. med. TVissensch., Berlin, 1869, vol. vii. S. 530.

2 "The Spectroscope in Medicine," London, 1880, p. 158.

^ Journ. Physiol., Cambridge and London, 1885, vol. vi. p. 24.

* Bogomoloff, loc. eit.; Heynsius and Campbell, Arch. f. d. ges. Physiol., Bonn, 1871,
Bd. iv. S. 540.

* Gamgee, " Phj'siological Chemistry," London, 1893, vo]. ii. p. 333; sec also Hoppe-
Seyler, loc. cit.

*• Loc. cit., p. 27.



REABSORPTION OF BILE SAL TS. 391

CJiolesterin. — The amount of cholesterin in bile is very variable,
ranging from 0*5 to 5 per cent. Cholesterin is insoluble in water or
dilute saline solution, and is dissolved in bile by the agency of the bile
salts, in solutions of which it is easily soluble. When the amount of bile
salts is insufficient to hold it in solution, it slowly passes out of solution
in a concretionary form around any particle of foreign matter present in
the bile, or around an existing concretion forming in this manner a
variety of gall stone.

According to Hoppe-Seyler, cholesterin is a cleavage product,
constantly formed in the metabolic changes of the living cell ; and for
this reason it is that cholesterin is invariably found as a chemical
constituent of both animal and vegetable cells. Cholesterin does not
easily undergo decomposition in the animal organism when once formed,
and is principally excreted in the higher animals in the bile. It is
found in increased quantity in tissue which is undergoing pathological
change ; this may, perhaps, be due to increased inability on the part of
the cells in their vitiated condition to break up the stable cholesterin.
Cholesterin is found in largest quantity as a constituent of the myelin
of nerve fibres and in the blood corpuscles. It is probably formed most
in the metabolism of nerve tissue, taken up by the liver cells from the
blood, and passed as an excretion into the bile ducts.

Cholesterin is purely an excretion, and is not reabsorbed, but passes
out of the body with the faeces. This is also the fate of the bile
pigments, which are gradually reduced to hydrobilirubin (stercobihn)
in their passage along the intestine. This substance may easily be
extracted from the faeces by absolute alcohol, after making acid with
sulphuric acid. The bile pigments have a poisonous action when
injected into a vein, which indicates that if they are reabsorbed at all
they must be changed in the process.^

Lecithin. — The amount of lecithin present in bile is much greater
than in any of the other secretions. All the lecithin, and any direct
products of its decomposition to be removed from the body, are carried
off in the bile. As lecithin, as well as cholesterin, is one of the con-
stituents of nerve tissue, the liver, by means of the bile, may be looked
upon as the great channel for the removal of the products of nervous
metabolism. Lecithin is also held in solution by the bile salts.

Reabsorption of bile salts — Their functions in the organism. —
The bile salts differ from the other bihary constituents in that they are
not purely an excretion. They are to a large extent reabsorbed, and
undergo a circulation in the body, with the probable function of acting
as carriers for the otherwise insoluble cholesterin in the bile. Such an
absorption of bile salts has been shown to take place in different ways,
which are, briefly, as follows : —

1. Bile salts taken by the mouth, cause an increased flow of bile ;
indeed, from recent observations by various experimenters,^ it seems that
the bile salts are the only substances which truly act as cholalogues.
This action can only be due to their absorption followed by an increased
elimination of them by the liver.

2. The bile of the dog contains only taurocholates. If unpau'ed

1 De Bruin, Centralhl. f. klin. Med., Bonn, 1890_, Bd. xi. S. 491.

^ Baldi, Arch. itcd. de bioL, Turin, 1883, tome iii. p. 395 ; Paschkis, Schmidt's Jahrh.,
Leipzig, 1885, Bd. ccvi. S. 19; Nissen, Centralhl. f. d. med. IVissensch,, Berlin, 1890, Bd.
xxviii. S. 948.



392 CHEMISTRY OF THE DIGESTIVE PROCESSES.

cholalic acid be given by the mouth, the amount of bile is increased, but
still only taurocholates are found in the bile ; but Weiss ^ found, after
giving sodium glycocholate for three days (5-9 grms. per diem), that
the bile in the gall bladder at death contained giycocholates, amounting
to 25-30 per cent, of the total bile salts present.

3. No connection exists between the amount of proteid metabolism
and the amount of cholates produced, such as would be found if the
cholates were a channel for the excretion of the nitrogen and sulphur of
proteid decomposition products.^

4. Tappeiner ^ identified bile salts in chyle obtained from the
thoracic duct in the dog.

5. Bidder and Schmidt * only found cholalic acid in traces in the faeces.
A review of all these facts shows that the bile salts are not an

excretion, but perform a circulation in the body. Besides the function
of dissolving the cholesterin to be excreted, the bile salts are also
credited with the effect produced by bile in aiding the absorption of
fats.^ Again, bile salts dissolve insoluble soaps of the alkahne earths.
This may be shown by precipitating a soluble soap with calcium or
magnesium sulphate, and then adding a solution of bile salts and gently
warming when the precipitate dissolves.'' Maly and Emich state that



Online LibraryE. A. (Edward Albert) Sharpey-SchäferText-book of physiology; (Volume v.1) → online text (page 55 of 147)