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

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till it has a deep orange colour, and is poured into at least an equal volume of
ether, when an amorphous brown precipitate falls, consisting of the greater
portion of the pigment present, in almost pure condition. The precipitate
may be filtered off, dried on the paper, and washed with a little chloroform
and absolute alcohol.-

Properties. — The substance so prepared is a h3^groscopic, brown,
amorphous substance, easily soluble in water, nuich less soluble in
alcohol ; only shghtly soluble in acetic ether, amyl alcohol, or acetone ;
and insoluble in chloroform, ether, and benzene. Its solutions show no
definite spectroscopic absorption-bands, even after the addition of acids.
Zinc chloride and ammonia produce no liuorescence. Alkalies give the
solution a brownish tint, acids a reddish brown. The pigment forms
insoluble compounds with the heavy metals, and is precipitated by
phosphotungstic and phosphomolybdic acids. With strong nitric acid
it undergoes a colour change resembling the xantho-proteic reaction.

Urochrome we have seen to be a pigment which can be removed
from the urine without the use of strong reagents, and in the removal
of which the fluid loses nearly all its colour. At the same time, its aqueous

^Proc. Eoy. Soc. London, 1894, vol. Iv. p. 394.

^ See also Kramm, Deutsche med. Wclinschr., Leipzig, 1896, Bd. xxii. S. 25 and 42. This
author separates the pigment by an entirely different method. He confirms Garrod's
original account of its properties.


solutions have a tint like that of urine itself, and, like normal urine,
show no absorption-bands. There can be no doubt, therefore, that it is
the essential cause of normal urinary coloration.

Physiological relations. — Until quite recently, we had no knowledge
of the chemical relationship, or of the metabolic precursors of this im-
portant physiological pigment. But Eiva ^ and Chiodera ^ have obtained,
by the action of potassium permanganate upon solutions of urobilin,
a substance which they believe to be identical with urochrome. A. E.
Garrod^ has added still more conclusive evidence for the existence
of a simple relation between this pigment and urobilin, by observing
that alcoholic solutions of pure urochrome, when treated with aldehyde
(which we may believe acts as a mild reducing agent), yields a pigment
showing the spectrum and all the more characteristic properties of
urobilin. The establishment of this relation is most important in
bringing our knowledge of physiological pigments into line, since, as
will be shown immediately, the derivation of urobilin from blood and
bile pigments is clearly established. We can now ascribe a similar
orig-in to the fundamental colouring matter of urine.

(b) Urobilin. — In 1868, Jaffe,* as an outcome of a spectroscopic
study of the urine, discovered a pigment with well-characterised pro-
perties, to which he gave the name of urobilin.

This pigment is perhaps scarcely entitled to be classified among
the preformed pigments of normal urine, for it is present as a rule in
minimal amount and almost always in the form of a chromogen. But on
rare occasions the free pigment is found in the fresh urine of normal
individuals, and, moreover, the importance of urobilin in other respects
makes it necessary to give it a prominent place in this section. It was
the first physiological urinary pigment of which we had accurate know-
ledge from the point of view of genesis and metabolic history. Its
increase in disease is a familiar phenomenon. These facts and its well-
marked spectroscopic characters have made it predominant in the
literature of urinary pigments. Even at the present time it is some-
times described as the essential colouring matter of urine, an error
which is at once demonstrated if the spectroscopic indications of normal
urine and of weak solutions of pure urobilin are compared.

Sejiaration. — When zinc chloride and ammonia are added to urine in due
proportion, a precipitate is obtained (cf. " Separation of Creatinin," p. 599), which
contains much of the lU'obiKn present. This method of precipitation was used
by Jaffe, and from the zinc precipitate he succeeded in extracting the pigment
in a remarkably pure condition, but in small quantity, and by a somewhat
complicated procedure.

Mehu ^ later showed that saturation of the urine with ammonium sulphate,
after acidification with weak sulphuric acid, produced a complete precipitation
of this pigment. The precipitate thus produced, which mainly consists of
pigmented lu-ates, will yield to acid alcohol a solution, in which the character-
istic absorption-hand of acid urobilin, to be later described, is easily seen.
Even when normal urine has been employed, the spectrum may be observed
after this procedure, for the bandless chromogen is decomposed by the acid

1 Gazz. med. di Torino, 1896, vol. xlvii. No. 12.
"^ Arch. ital. di din. med., Milan, 1896, vol. xxxv. p. 505.
^ Journ. Physiol., Cambridge and London, 1897, vol. xxi. p. 190.

^ Centralhl. f. d. med. JVissensch., Berlin, 1868, Bd. vi. S. 243 ; Firchow's Archiv, 1869,
Bd. xlvii. S. 405.

^ Bull. Acad, de med., Paris, 1878, tome vii. p. 671.


employed. When a urine rich in urobilin is saturated with aninioniurn
sulphate (best after previous removal of the urates by preliminary saturation
with chloride of ammonium), and acidified with sulphuric acid, it will yield
the pigment when shaken with a mixture of ether and chloroform. From this
organic solvent distilled water will again remove all the urobilin, and from
the water it may be precipitated by the further use of ammonium sulphate.
A method of separation may be based upon these facts which will yield a
very pure product in comparatively large amount.^

Properties. — Urobilin is an extremely soluble substance, dissolving
freely in all ordinary solvents. It is, however, proportionately less
soluble in water than is urochrome, though much more readily soluble
than the latter in alcohol and other organic liquids. Its solutions, when
concentrated, have a brown colour ; when more dilute they are yellow ;
on great dilution they exhibit a highly characteristic change to a dull
pink colour.

An alcoholic solution of the pure pigment free from extraneous acid
or alkali exhibits a green fluorescence quite apart from the addition of
reagents. When, however, zinc chloride and ammonia are added, a
greatly increased fluorescence is produced. This striking reaction is of
much value in the identification of urobilin ; it may be obtained after
great dilution.

Solutions of urobilin exhibit very definite spectroscopic phenomena.
In clear acid solutions of moderate strength, a single absorption-band is
seen between the Fraunhofer lines h and E, slightly overlapping the
latter; situate, therefore, at the junction of the green and blue of the
spectrum (Fig. 57, Spectrum 4). In highly concentrated solution this band
is lost in a general absorption of the more refrangible rays. On diluting
such a concentrated solution a broad band first appears with a region of
complete blackness towards red, and a dark shading towards violet. As
dilution proceeds the shading first disappears, and then the dark portion
of the band shrinks till its limits extend from about \ 508 to a 486.
After this the width of the band is constant, until with very large
dilution it grows faint and ultimately disappears (Fig. 57, Spectrum 5).
The activity of the pigment in absorbing light in this region is enormous,
and a solution so dilute as to have a very faint colour indeed, will show
a well-marked band. An absorption-band of an intensity such as is
occasionally seen in normal urine, would correspond to that of an
almost colourless solution of the pure substance.

Urobilin, like most animal pigments, shows acidic tendencies, and
forms compounds with bases, being liberated from these combinations
on the addition of an acid.

If ammonia be added to a solution of the free pigment, the colour
changes to a canary -yellow, and unless the solution be very strong the
absorption-band disappears. The sodium and potassium compounds
have a colour in solution more like that of the free pigment, and show
an analogous band, which is situate, however, somewhat nearer the red.
The zinc compound in ammoniacal solution fluoresces, as we have
already stated, and shows with the spectroscope a band almost identical
with that of the potassium and sodium compounds. The calcium com-
pound is yellow in solution and shows no band. Mercury forms a pink
compound, with a band nearer to the red than any of those pre\dously
referred to. A solution of mercuric chloride will develop a pink colour

^ Garrod and Hojikiiis, Journ. FhysioL, Cambridge and London, 1896, vol. x.\. p. 120.


when a]Dpliecl to tissues stained witli urobilin, and may thus be used as
a test for such staining (Adolf Schmidt).

When to a concentrated solution of nearly pure urobihn in sodic or
potassic hydrate, sufficient sulphuric or hydrochloric acid is added to
render the liquid faintly acid, a sHght turbidity is oljserved, due to the
liberation of the free pigment from its more soluble alkahne combination.
If the turbid liquid be examined with the spectroscope, there is seen, in
addition to the ordinary acid band between I and F, a sharply-defined
narrow band in the green, enclosing, and being almost bisected by the
Fraunhofer line E (Fig. 57, Spectrum 6). This extra band is most probably
due to the special light absorption exercised by the impalpable particles of
solid urobilin in suspension. It wholly disappears when the precijitate
is filtered off, or when it is redissolved, the ordinary band alone laeing
then visible.^

Solid urobilin is an amorphous red-brown substance, which, when
isolated and dry, may be kept without decomposition. It is not
deliquescent, but fuses at comparatively low temperatures, afterwards
sohdifying to a brittle transparent shellac-Hke form. It has a slight
but peculiar and characteristic odour.

Physiological relations. — Urinary urobilin is identical with the chief
pigment of fseces (stercobilin). So certain is the identity of these two
substances, that it is undesirable to retain separate names for them.

Urobilin is closely related to the pigments of the bile. This was
from the first recognised by Jaffe ; and shortly after the discovery of
the pigment, Maly prepared a substance (hydrobilirubin) by the
reduction of Ijilirubin with sodium amalgam, which he held to be
identical with urobilin. That the urinary pigment is a reduction pro-
duct of bilirubin is likely, but it is probable that hydrobilirubin, as
described by Maly, represents an intermediate stage in the reduction.
It differs at any rate somewhat from urobilin as it occurs naturally.

Urobilin is formed, however, when bile decomposes out of contact
with the air, and it may be extracted from the bile removed post-
mortem from the gall bladder.

Several observers have shown that intestinal micro-organisms can
effect the reduction of bilirubin to m'obilin.

This pigment, or substances closely allied to it, can be prepared direct from
haemoglobin derivatives — heematm and haematoporphyrin — by reduction pro-
cesses. It has been stated that oxidation is also capable of yielding urobilin
from bile and blood pigments respectively, but it is not conceivable that
both reduction and oxidation could lead to the same chemical result, and there
is in this matter an anomaly which requires explanation. It must not he
forgotten that peroxides (peroxide of hydrogen and peroxide of lead, have
been employed in this connection) may in a sense act as reducing agents, free
oxygen being given off by the interaction of the peroxide and any easily
reducible compound with which it is brought in contact.

Urinary urobilin has not yet been analysed. If the formula of
hydrobihrubin lie compared with those of the related pigments, it will
be seen that both reduction and hydration probably occur in its

Haematin . . . C.oHgaN^O^Fe
Bilirubin . . . CooHggN^Oo
Hydrobilirubin . . Co^H^qN^O-
1 Garrof] and Hopkins, Journ. Physiol., Cambridge and London, 189C, vol, .\x. p. 125.


The change from biliruljiu to hydrobilirubin may be thus ex-
pressed —

If urobilm differs from hydrobihrubin, the difference is possibly,
as ah^eady stated, in the direction of increased reduction.

The origin of urinary urobilin is probably threefold — from absorp-
tion of the ready-formed pigment in the Ijowel ; from direct production
in the liver ; and, lastly, from reduction of the blood pigment in the
organs, independently of hepatic agency.

Of the precise nature of the chromogen of urobilin we have no
knowledge. It is precipitated intact when normal urine is saturated
with ammonium sulphate in the absence of mineral acid.^ It is possible
that oxidation may decompose it, as some urines originally showing
no absorption-band will develop such on standing. This phenomenon
might follow, however, from the decomposition during standing of
some compound of the pigment with lime or other base.

(c) Uroerythrin. — This pigment is best known as the colouring
matter of pink urate deposits. It is a substance of the greatest interest,
but one which has proved, from its marked instability, elusive and
difficult of investigation.

It was first dealt with as far back as 1800, by Louis Proust,
under the name of acicle rosacique. Its present name was assigned
to it by F. Simon in 1850 — the term "purpurin," earlier proposed by
Golding Bird, being still sometimes used. Heller published an account
of the pigment in 1854, and Macmunn first accurately described its
spectrum in 1883. Very important contributions to our knowledge of
uroerythrin have recently been made by Eiva, Zoja, and A. E. Garrod.^

The quantity of the pigment excreted is, under any circumstances,
very small; but its tinctorial power is extremely high, and when in
solution it may materially contribute to the coloration of the urine.
It is certainly to be looked upon as a pigment of normal urine, as
urates coloured by it frequently separate from the excretion of persons
in health.

Separation. — A quantity of pink urate deposit is collected upon a filter,
Avashed with ice-cold water, dried, and soaked in absolute alcohol. The
alcohol, though a solvent for uroerythrin, does not extract it from the urates.
The spirit is poured off and the precipitate dissolved in warm water ; from the
aqueous solution so obtained the pigment is easily and completely extracted by
shaking with amylic alcohol (Riva). Garrod has shown that if the pink urates
are first dissolved in warm water, and are then reprecipitated by saturation
with ammonium chloride, the pigment is carried down with them afresh, and
in such a condition that it may now he extracted with alcohol. An alcoholic
solution, if diluted with water, may he washed by shaking with neutral
chloroform, which removes impurities hut no uroerythrin. But if after this
preliminary washing a fresh supply of chloroform is added, together with a
single drop of acetic acid, on shaking, the pigment is now found to be transferred
completely to the chloroform as an effect of the acidification of the liquid.

Properties. — The most striking properties of uroerythrin are — (1) Its
remarkable affinity for uric acid compounds ; (2) the ease with which

^ Eieholz, .Tourn. Physiol., Cambridge and London, vol. xiv. p. 326.
- Journ. Physiol., Cambridge and London, 1895, voL xvii. p. 439. Full references to
the literature will here be found.



its solutions are decolorised by light ; and (3) its colour reactions
with the caustic alkalies and mineral acids. The pigment invariably
associates itself with urates during their precipitation ; either when they
separate naturally from a urine containing it, or when they are arti-
ficially added to its pure solutions, and are allowed afterwards to separate.

E. b. F.

Fig. 57. — Chart of spectra.

1. Acid haematopoiijhyrin.

2. Alkaline hseniatopoiphyrin.

3. HEematoporphyrin as found in urate sediments.

4. Acid urobilin — concentrated.

5. Acid urobilin — dilute.

6. The E band spectrum.

7. Uroerythrin.

8. Urorosein concentrated — on dilution the band

shrank rapidly from redward end.

It apparently forms a loose compound with the urates, as a special
absorption-spectrum is seen when light passes through the pink pre-
cipitate, differing from that proper to solutions of the pigment (Garrod).
The best solvent of uroerythrin is amylic alcohol ; acetic ether is but
little inferior, and the pigment is also soluble in alcohol, chloroform, and


water. The solutions have a rich orange colour ; only when very dilute
and quite free from impurity do they exhibit a pink tint. All solutions
of the pigment are decolorised on exposure to light, even to subdued
daylight. On the other hand, light has little effect upon the solid
pigment, and none at all upon pink urate sediments.

When solid uroerythrin is treated v^ith solutions of the caustic
alkalies, a remarkable green coloration is produced (Thudichum). Green
derivatives from animal pigments are so uncommon that the reaction is
highly characteristic. It can be well seen when a little pink urate
deposit is collected upon a filter, dried, and then touched with a drop of
sodium-hydrate solution. If a solution of the pigment be treated with the
same reagent, a rapid play of colours may frequently be seen, from pink,
through purple and blue, to grass-green. With acids, colour-reactions
also occur, but they are somewhat less certain, being dependent upon
exact conditions of experiment. If to a solution of the pigment
sulphuric acid be added, the deep orange colour changes to a brilliant
carmine. Hydrochloric acid produces a rose-pink, phosphoric acid a

Examined spectroscopically, a solution of uroerythrin, at a suitable
degree of dilution, shows two somewhat ill-defined absorption-bands
united by a shading of less intensity (Macmunn). The more red-ward of
these is seen in the green between the lines D and E, and nearer the
latter ; the other closely agrees in position with the ordinary urobilin
band at F (Fig. 57, Spectum 7). Pink urate sediments and the carmine
derivative produced by sulphuric acid agree in giving a single banded
spectrum, namely, a broad band extending from the D line towards violet.

(d) Hsematoporphyrin. — In 1881, Neusser^ and Macmunn^ observed
the occurrence in urine of pigments closely related to heematoporphyrin.
During the following decade the work of le Nobel, Stockvis, Salkowski,
Hammarsten, Copeman, and others extended this discovery, and it
became established that hsematoporphyrin itself is a constituent of
certain pathological urines. In 1892, A. E. Garrod^ showed that it
is also to be found in normal urine.

In health the pigment is excreted in very small amount, and can
scarcely be said to function as an active colouring matter of the urine ;
but it is of the highest interest to recognise that this iron-free deriva-
tive of haematin, which in the laboratory is only to be obtained by the
use of strong reagents, is a normal physiological product. In patho-
logical conditions, and especially after the use of certain drugs, it is
present in greatly increased amount.

Isolation from normal urine. — The method recommended by Garrod de-
pends upon the fact that the pigment is carried down by the precipitate of
phosphates produced on the addition of caustic alkah to the urine. After
special treatment of this precipitate, the pigment may be obtained in chloroform
solution. The chloroform is evaporated, and the residue washed Avith neutral
alcohol and dissolved in acidified alcohol, when a sohition is obtained of pure
pink colour, comparable with solutions of the purest specimens of the pigment
obtained from blood, and showing the spectrum of acid hsematoporphyrin with

^ Sitzungsb. d. k. Akacl. cl. JVissensch., Wien, 1881, Bd. Ixxxiv. S. 536.
" Pj'Oc. Roy. Soc. London, vol. xxxi. p. 206.

^ Journ. Physiol, Cambridge and London, 1892, vol. xiii. p. 598 ; ibid., 1894, vol. xvii.
p. 349.

VOL. I. — 40


Pathological urines rich in the pigment will generally yield, it easily to
acetic ether and to amylic alcohol.

Properties. — An account of the properties of hfematoporphyrin will
be found in the section devoted to blood pigments ; but the pigment as
found in the urine has certain peculiarities which must be referred to here.

When the urine is sufficiently rich in the pigment for the absorption-
bands to be visible without treatment (always a pathological condition),
it is found that the bands observed are those of the so-called alkaline
hffimatoporphyrin (Fig. 57, Spectrum 2). Indeed, if a solution of the
pigment showing the acid spectrum (but, of course, free from excess of
mineral acid) be added to urine, the bands are seen to change to those
of the alkaline form, even though the urine itself be of normal acidity.
Acid sodium phosphate will, in fact, yield base to the haematoporphyrin,
unless, indeed, the salt is in great excess, when it can, on the other
hand, convert the alkaline form of the pigment into the acid. These
facts form an interesting commentary on what we have said in the
section devoted to the acidity of the urine, as to the complex conditions
which govern the phenomena of chemical reaction in the fluid.

Urinary hsematoporphyrin may be in the form of unstable modi-
fications. Alkahne solutions of the pigment obtained from many
specimens exhibit a' five-banded instead of a four-banded spectrum
(Macmuun). Occasionally, too, urate sediments may be pigmented with
a form of the pigment which, in alkaline or neutral solution, shows a
spectrum of two bands resembling that of oxyhsemoglobin (Fig. 57,
Spectrum 3). Dilute mineral acids, however, promptly change this spec-
trum to that of ordinary acid hfematoporphyrin (Fig. 57, Spectrum 1).
There is some evidence that a colourless chromogenic substance, related
to hfematoporphyrin, may occur in the urine, as the pigment has been
observed to increase in amount after standing.

Chromogenic substances in urine. — Two, at least, of the pig-
ments we have now described (urobilin and haematoporphyrin) may
exist, as we have seen, in the form of chromogens — colourless, or less
coloured, precursors. But the urine contains other chromogenic sub-
stances, which in the original urine always, or nearly always, retain
their colourless form ; and, as a rule, take no share in the true
pigmentation of the fluid.

We do not include, under the term of " chromogen," all substances Avhich,
by the action of strong reagents, happen to be capable of yielding a coloured

We purposely exclude such bodies as the so-called " humous substances" of
Udransky — indefinite products of wholly doubtful nature — obtained by such
processes as fusing urinary precipitates with caustic alkali, or boiling the
previously concentrated urine for hours with hydrochloric acid. These are
probably derived from the carbohydrates and other constituents of the urine,
by the destructive action of the reagents. Beyond the fact that they happen
to be amorphous, and yellow or brown in colour, there is nothing to suggest
that they are related to xu'ochrome or any other definite pigment.

We shall deal oidy with those chiomogenic substances which are of
importance, either because they may, though with great rarity, appear
as actual pigments, or because they yield their coloured derivatives
with comparative ease, and may thus lead to confusion when the urine
is beins investigated in other connections.


(a) Indoxyl (indigo-blue and indigo-red). — Incloxyl (cf. pp. 607
and 631) easily oxidises to indigo -blue, or to the isomeric substance
indigo-red. The relation between indoxyl and its blue derivative is
expressed by the following equation : —

C.H<Tn^/™ + 0' = C.H/gg>C = 0<^g)cA + 2H,0

(indoxyl) (indigo-blue)

The formula of indigo-red is C6H,<^-^^^)C = C<^^1^^^^N, and it

arises, like its blue isomer, when, by oxidation, four atoms of hydrogen
are removed from two molecules of indoxyl. Oxidising reagents when
added to urine may, according to the conditions of the experiment, give
rise to the formation of either or both of these coloured derivatives.
The blue substance, however, is more easily and more generally

Online LibraryE. A. (Edward Albert) Sharpey-SchäferText-book of physiology; (Volume v.1) → online text (page 88 of 147)