“ On the frequent occurrence of Indigo in Human Urine, and on
its Chemical and Physiological Relations.” By Arthur Hill
Hassall, M. D. Physician to the Royal Free Hospital, &c.
[The following is an abstract of a very interesting paper, by
Dr. A. H. Hassall, read at one of the late meetings of the Roy-
al Society, London. Its publication may, perhaps, induce others
to repeat Dr. H.’s investigations, with the object of defining
more especially than he has done, the diseases in which the blue
indigo occurs, so that the discovery may be applied to a practical
purpose in diagnosis.]	S. L.
The author was led to the investigations laid before the So-
ciety in the above communication, by the following circum-
stances :—
Some three or four years since, when examining urinary de-
posits under the microscope, he frequently observed in the field
of vision, particles of a deep blue color; so often did this occur
that he could not even then help suspecting that their presence
was not accidental; however, no analysis of the blue coloring
matter was made at the time, and the circumstance was in a fair
way of being forgotten, until the recollection of it was revived
by another occurrence.
In June 1852, a sample of urine, freely exposed to the air in
an open vessel, was observed after four or five days, exposure,
gradually to change color ; tjie pellicle or scum which had formed
upon the surface of the urine became first slate-color, and at
length deep blue, with here and there a rust-red tint. The urine
also underwent at the same time some remarkable changes; it
became thick and turbid, deep brown, greenish, bluish-green, and
finally of a faded yellowish-green color ; a considerable sediment
was found at the bottom of the glass; this was deep brown, inter-
mixed with a little blue coloring matter, and it had a medicinal
smell resembling somewhat that of Valerian. In this state,
without undergoing any further material changes, the urine re-
mained for many days.
Examined with the microscope, the scum or pellicle on the sur-
face was found to consist of vibriones, innumerable animalcules,
and crystals of triple phosphate, with a great many fragments
and granules of a deep and bright blue color.
A second sample of the same urine was therefore procured,
every precaution being taking to avoid fallacy. Gradually the same
changes ensued as in the first sample, and this likewise became
blue. Having thus ascertained that the changes observed were
due to something contained in the urine itself, the author next
proceeded to set aside in open vessels a series of urines all from
the same patient, noting the alterations which occurred from day
to day; these samples underwent nearly similar changes, but the
quantity of blue coloring matter and brown extractive gradually
diminished, until at length they were present in such small
amount as to be visible only under the microscope, and at last
they entirely disappeared.
The result obtained from an examination of the urine, the blue
coloring matter and the brown extractive, are then given by the
author; they are as follow :—
The urine.—The urine of the second sample at the time of
analysis, when shaken, had a dark greenish-brown color; but on
standing at rest for some time, the coloring matter fell to the bot-
tom, forming bluish-green flocculi, while the supernatant liquid
was of a deep wine-red color. The bottle was set aside corked,
for ten days, at the end of which time the bluish-green precipi-
tate had entirely disappeared ; but on removing the cork and
allowing free access of air for some time, the colored deposit was
again produced. This was washed with water, drenched with
hydrochloric acid, and finally dried ; by this means was obtained
a rich blue powder possessing all the chemical characters and
properties of indigo.
The urine that was filtered off from the above precipitate was
allowed to evaporate spontaneously, by which means it yielded
an additional quantity, which adhered in the form of very small
flakes to the sides of the dish. It also gave a rather large pro-
portion of a deliquescent brown coloring matter, which was
treated, first with alcohol, and then with water.
The alcohol acquired a deep brownish-red color, and the water
a dark brownish-red green. Both of these solutions were evapo-
rated at a temperature of 160® Fahr. The alcoholic solution
furnished a rich brown extractive which was soluble in water, but
not in dilute acids, and nitric acid did not produce that play of
colors which is characteristic of bile-pigment; nor did the precip-
itate formed with basic acetate of lead furnish a purple liquid
with alcohol and free acid. A strong solution of potash dissolved
the extractive and yielded a deep blood-red fluid, which was ren-
dered green and opalescent by boiling. These reactions show
that the brown pigment was somewhat like haematine in its
chemical manifestations.
While the aqueous solution of the brown matter was undergo-
ing evaporation, it gave a further supply of indigo, which was
formed most freely at the edge of the liquid. The residue was
made black by concentrated sulphuric acid, and deep brown by
potash.
The blue coloring matter.-—Of two samples of this in a dry
state, mixed with a large quantity of earthy phosphates, vibri-
ones, mucus, and epithelium, one gave a dark brown solution with
concentrated sulphuric acid, and the other a dirty blue. Both
of these solutions were decomposed by water, furnishing in the
former case a dark brown deposit, and in the latter a dirty green.
In most of their other reactions, however, they presented the
characters of indigo ; and it is especially deserving of notice, that
they were reduced by lime and sugar, giving a liquid from which
hvdrochloric acid threw down a greenish-blue precipitate.*
*The cause of concentrated sulphuric acid giving with one of these sam-
ples a brown solution, and with the other only a dirty blue, was, the author
considers, mainly owing to the large quantity of animal matter with which
the specimens were contaminated ; the acid from its charring effect on this,
would produce a brown, or blackish solution, thus obscuring the color of the
solution of indigo.
The brown extractive.—The brown extractive yielded nearly
the same results as on its first analysis, and the aqueous solution
furnished a few blue flocculi. A portion of the alcoholic extract
was heated with Liq. Potassse for the purpose of ascertaining
whether it contained leucine, and the product, on being treated
with hydrochloric acid, gave off a powerful odor, which was some-
what like valerianic acid ; but the result was too doubtful to be
of much value. The author had already referred to the peculiar
smell of Valerian emitted by the extractive of more than one of
the samples. He considers that the clearest and most positive
evidence was thus obtained that the blue coloring matter in this
case was indigo.
It was not very long after the occurrence of the first case of
blue urine that numerous other instances fell under the author’s
observation. The urines of all these cases underwent very nearly
the same changes as in the first; in some the quantity of blue color-
ing matter found was very considerable, in others less ; and in
the third class of cases the microscope was necessary for its dis-
covery. In nearly all these cases the blue coloring matter was
submitted to analysis and ascertained, on the clearest evidence,
to be indigo.
The author in the next place considers the question of the
source and origin of indigo in the urine.
It appeared that in the cases related by the author, colored in-
digo was not present in the urine when first voided, but that it
was gradually formed some time afterwards by a process of oxi-
dation or exposure to the air, being in most of the cases probably
derived from the brown extractive, which in its chemical mani-
festations so closely resembles hsematine.
The author contrasts cyanourine with the indigo detected in
mine. He observes that the most distinctive tests laid down for
cyanourine are its solubility in boiling alcohol, and the action of
sulphuric acid upon it, which gives a reddish-brown solution ; and
states that he had ascertained that these tests are not to be re-
lied upon, since indigo, when contaminated (as in the urine it
frequently is) with a large quantity of animal matter, vibriones,
&c., gives a reddish-brown solution with concentrated sulphuric
acid, from the charring of the animal matter, and in many cases
forms a bright blue solution with boiling absolute alcohol; hence
he could not help suspecting that cyanourine and indigo are very
closely connected with each other, if they be not identical. He
observes, it is at least singular that while so many cases of indi-
go were met with, not a single instance of cyanourine presented
itself. He also contrasts indigo with apoglancine, and remarks
that this is acknowledged by Heller himself to be nothing more
than cyanourine mixed with urrliodine.
Take then into consideration the whole of the facts described
in this communication, and the followiug conclusions are deducted:
1st. That blue indigo is frequently formed in human urine,
the quantity being subject to the greatest variation; in some
cases it is so considerable as to impart a deep green, or bluish-
green color to the whole urine ; a pellicle of nearly pure indigo
also extending over the entire surface of the liquid ; while in others
it is so small that it can only be detected by means of the micro-
scope.
2d. That for the formation of this indigo, it is in general
necessary that the urine should be exposed to the air for some
days in an open vessel, oxygen being absorbed and the blue in-
digo developed. Whatever, therefore, facilitates oxygenation, a
free exposure to light and air, warmth and sunshine, hastens the
appearance of the indigo; hence in summer the changes described
take place much more quickly than in winter; on the contrary,
the changes are retarded and even stayed by the exclusion of the
atmosphere. Blue indigo may even be deprived of its color and
reformed, alternately, according as air is excluded or admitted to
urine containing it. Brom some of the cases recorded, it would
appear, however, that blue indigo is occasionally formed in the
system, and is voided as such in the urine.
3d. That there is usually found with the blue indigo, where the
amount of this is considerable, a brown extractive, sometimes in
large quantity, the aqueous solution of which, by exposure to air,
yields a further supply of colored indigo, and which closely resem-
bles haematine in its chemical manifestations and elementary com-
position. There is therefore great reason for believing that in the
majority of the cases here recorded, the blue indigo was derived
from altered haematine, although it is at the same time probable,
that in some cases it is formed from modified urine pigment,
which is itself supposed to be a modification of haematine. Be-
tween the greater number of the animal coloring matters there
is the closest relationship in chemical composition, so that the
transformation of the one into the other would appear to be both
easy and natural.
4th. That the urines in which the colored indigo occurs in the
largest quantity, are usually of a pale straw color, readily becom-
ing turbid, alkaline, and of low specific gravity. Small quanti-
ties of indigo are however found in urines possessing characters
the very reverse, that is, in such as are high-colored, acid, and
of high specific gravity ; but as a rule, in these urines the blue
pigment is usually absent.
5thly. That as colored indigo does not occur in healthy urine,
and since, where the amount of this is at all considerable, it is
accompanied with strongly-marked symptoms of deranged healthy
the promotion of blue indigo in urine, must be regarded as a
strictly pathological phenomenon, apparently associated rather
with some general morbid condition, than essentially with disease
of any one organ; although there is reason for believing that
the blue deposit is met with very frequently in Bright’s disease,
and in affections of the organs of respiration, it should however
be remarked that none of the worst cases of indigo in the urine
which the author met with, were cases of Bright’s disease.
The paper is illustrated by drawings, and a specimen of the
indigo, as deposited from urine, was exhibited.—Philosophical
Magazine, Sept, 1853.