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obtained.

It is of great rarity for the urine to be actually pigmented
with indigo-blue. As we have already seen (p. 607), the urinary
indoxyl is excreted in the form of a conjugated sulphate, and
this compound ■ resists oxidation. Only when the indoxyl is first
liberated from its combination does the action of oxidising reagents
produce the blue colour. It is stated, however, that the urine of
cholera may sometimes exhibit a blue shade from the presence of
indigo-blue. We have seen that the amount of indoxjd sulphate is
increased in the urine whenever bacterial putrefaction of albuminous
substances is occurring to a greater extent than usual, whether in the
bowel or elsewhere in the body (putrid abscesses, etc.). The most
ready method of demonstrating the amount of indoxyl is by converting
it into indigo-blue. Jaffa's test} — The urine is mixed with an equal
bulk of strong hydrochloric acid, by which means the "indican"
(indoxyl sulphate) is decomposed and the indoxyl liberated. With a
pipette, a solution of a hypochlorite is now added to the mixture drop
by drop, when, by oxidation of the indoxyl, indigo-blue is formed. By
shaking up the liquid with chloroform, a solution of the blue substance
is obtained in the latter (Stockvis, Senator, and others). Otherwise,
a crystal of potassium chlorate is placed at the bottom of a test tube
and covered with the urine to be examined. Strong hydrochloric acid
is then allowed to run down the side of the tube so as to reach the
crystal without mixing with the urine. The latter floats upon the acid,
and at the junction of the fluids a blue ring is seen of intensity varying
with the amount of indoxyl present.

But indigo-blue is itself an easily oxidisable substance. It is
instantly decolorised by nitric acid, and without difficulty by hypo-
chlorites. In Jaffe's test, as above described, it is therefore necessary
to add the oxidising agent with great care, or the blue colour will
disappear as soon as formed. In Obermayer's- method, the urine is
first precipitated by acetate of lead, and filtered ; to the filtrate is added
an equal bulk of strong hydrochloric acid, containing two or three parts
per thousand of ferric chloride. The mixture is shaken for a short
time, and the liberated pigment taken up, as before, in chloroform. In

1 Arch.f. d. ges. Physiol., Bonn, 1870, Bd. iii. S. 448.

2 Wieii. Idin. JVchnschr., 1890, S. 176.



62 8 THE CHEMISTRY OF THE URINE.

this case the ferric salt acts as a mild oxidising agent, sufficient to form
but not to destroy the pigment.

With care a certain amount of indigo-blue may be obtained from
most normal urines ; and, apart from the increase in actual disease,
indoxyl may be present in considerable amount, and the urine yield
a well-marked indigo reaction, when nothing more than constipation
exists.^

Indigo-red is more liable to be formed from the urinary indoxyl
when Jaffe's test is applied with the aid of gentle heat. Higher
temperatures favour the formation of the red isomer, lower tempera-
tures the blue.^ In Weber's test for indicanuria both pigments are
formed. The urine is treated, as in other methods, with its own volume
of hydrochloric acid ; one to three drops of dilute nitric acid are then
added, and the mixture heated to boiling. After cooling it is shaken
with ether, when the urine, if rich in indoxyl, is found to retain a blue
colour, while the supernatant ether is red or violet. The formation of
indigo-red has no significance beyond such as is attached to that of
indigo-blue. It may sometimes arise from the urine on the addition of
strong hydrochloric acid alone {infra).

(b) Urorosein. — Quite distinct from indigo-red is the red pigment,
named " urorosein " by Nencki and Sieber,^ and since carefully studied
by H. Eosin.* It is produced from its chromogen by the action of
mineral acids ; best with the aid of an oxidising reagent, but frequently
appearing when the urine is treated with strong hydrochloric acid alone,
especially after standing. It is freely taken up, after its formation, by
amyl alcohol, but is not soluble in ether. Alkalies immediately destroy
its colour. The chromogen of urorosein is precipitated by saturation
with ammonium sulphate.^

(o) Skatoxyl-red, which is formed from skatoxyl on oxidation, is never
obtained from urine under ordinary circumstances (Rosin), though it may be
produced in the urine of animals when skatoxyl has been given by. the mouth
(Brieger).

It may be stated generally that when a red colour is produced in
urine by the addition of strong acids (with or without the assistance
of oxidising reagents), it will in the great majority of cases be due to
urorosein or to indigo-red. The two pigments may be easily distin-
guished, in that urorosein, unlike the indigo pigment, is not taken up
on shaking with ether or chloroform, and is easily decolorised by
alkalies.^

The Pigmentation of Pathological Ueines.

AU the pigments and chromogens that we have so far described may be
excreted in increased amount in disease. There are other pigments which
only appear in the urine pathologically.

In the urine of fever a well-marked band of urohihn may generally he
seen without preliminary treatment, and uroerythrin is often present in more

1 Cf. V. Jaksch, " Klinische Diagnostik," 1896, Aufl. 4, S. 406.

-Rosin, Virchow's Archiv, 1891, Bd. cxxiii. S. .519.

^ Journ. f. 2^rakt. Chem., Leipzig^ 1882, Bd. xxvi. S. 333.

* Deutsche mod. WchnscJir., Leipzig, 1893, S. 51.

'' Garrod and Hopkins, Journ. Physiol., Cambridge and London, 1896, voL xx. p. 134.

^ Rosin, Firchow's Archiv, 1891, Bd. cxxiii. S. 519.



PIGMENTATION OF PATHOLOGICAL URINES. 629

than normal amount. I have also frequently observed that urochrome itself
takes a share in the increased pigmentation of febrile urine.

Urobilin is found in large amount vyhen extensive haemolysis, or large
internal haemorrhages, have occurred ; it is also greatly increased in certain
cases of hepatic cirrhosis. The high colour of the urine of pernicious anaemia
is in part due to urobilin ; other pigments may take a large share in the increase
in colour, but it is characteristic of this disease for free urobilin to be present
instead of the chromogen, for even in pale specimens, which are sometimes
passed, an absorption-band between h and F is usually visible. It is common
for free urobilin to be present in diabetes. An increase of uroerythrin is
seen in many forms of hepatic disorder. Haematoporphyrin does not appear to
depend upon haemolysis for increased excretion. After excessive use of drugs
of the sulphonal type, the urine may exhibit a deep port-wine colour ; part,
but not the whole, of this pigmentation is due to an enormously increased
excretion of heematoporphyrin, which may be quite unassociated Avith any
decrease in the haemoglobin of the blood. Increase of this pigment, but of
much slighter degree, occurs also in plumbism and in certain other diseased
conditions.

The so-called pathological urobilin.— Several observers have made a
distinction between normal urobilin and a pathological form of the pigment.
The differences found have been mainly those of spectroscopic appearances ; the
pathological form showing a proportionately broader band between h and F,
and additional bands elsewhere. The various descriptions of the pathological
pigment are in no sense consistent one with the other. Evidence has recently
been brought forward to show that the points of distinction may be all
explained as the result of impurities, and that urobilin is one and the same
substance wherever found. ^

Special pathological pigments — Blood pigments. — In hcematuria, due
to whatever cause, the urine usually contains unaltered haemoglobin. In
general the pigment may be recognised in solution spectroscopically, while red
blood corpuscles are found in the deposit. Some specimens of urine preserve
the integrity of the corpuscles very completely, and in slight cases of haeuiaturia,
while no pigment may be found in solution, the deposit obtained by centrifuging
will show a red layer of corpuscles. In hiemo/jIoMmiria the pigment is passed
wholly in solution, and no corpuscles are found. Not infrequently methcerno-
glohin is present in place of or in addition to oxyhaemogiobin, even when
the urine is first passed. Specimens which are spoken of as " smoky " usually
contain this latter form of pigment.

If the quantity of pigment is too small to show a recognisable spectrum
direct, the urine may be heated with caustic alkali, filtered, and a few
drops of ammonium sulphide added. The more powerful absorption-bands
of haemochromogen will then be generally visible. Or, the urine may be
boiled with caustic alkali, when, in the presence of blood, a greenish tint is
produced, and the phosphates are precipitated with a brownish-red colour, due
to haematin (Heller). The blue colour produced by the addition of guaiacum
tincture and an ethereal solution of hydrogen peroxide, is a delicate but not
wholly conclusive test when applied to urine.

Bile jngments appear in the urine in most cases of jaundice, generally in
the form of bilirubin, when the urine is saflron-coloured ; but occasionally
partly as biliverdin, Avhen a greenish tint predominates. When present in
large amount, there is no difficulty in the recognition of these pigments.
Gmelin's reaction is obtained by allowing the urine to run gently on to the
surface of some fuming nitric acid contained in a test tube. The test is made
more delicate if the urine be first repeatedly filtered through a clean white

^ Hopkins, Guy's Hos]}. Rep., London, 1893, vol. 1. p. 363 ; Garrod and Hopkins, "The
unity of Urobilin," Journ^ Physiol., Cambridge and London, 1896, vol. xx. p. 130.



630 THE CHEMISTR Y OF THE URIAE.

filter paper ; the paper is stained yellow, and a drop of fnming nitric acid
allowed to fall upon it produces tlie characteristic play of colours. When
only traces of the pigment are present, Gnielin's test is best applied to the
precipitate produced in the urine by the addition of lime-water with the
subsequent passage of a stream of carbon dioxide ; the precipitate is filtered
off', dried, and touched with nitric acid.

Carboluria. — In poisoning by carbolic acid, and often to a less degree after
the substance has been freely used as a drug, the urine has a greenish-brown
or dark brown colour, which increases on exposure to the air. This colour is
due to oxidation jDroducts of some of the aromatic substances present in normal
urine, which have been dealt with on p. 607 et seq. They are excreted in much
greater quantity after the administration of phenol. Pyrocatechin and
hydrochinon are especially responsible for the colour elfect.

Alcajytomiria (cf. p. 607). — A phenomenon very similar to that present in
carboluria is seen in certain other conditions, where an alkaline urine, as it
stands in the air, takes first a brown colour at the surface, which gradually
spreads through the fluid, and may finally result in the whole urine becoming
nearly black. Such urine always reduces copper solutions. The phenomenon
was first observed by Boedecker in 1859, and it was later ascribed by him to a
substance which he called alcaptoji.

But alcapton, as aheady stated, is not a definite compound, and the colour
phenomena are probably due to the action of oxygen upon some of the
aromatic bodies present ; probably, at times, upon pyrocatechin and uroleucic
acid, but more often perhaps uj)on the homogentisic acid of Wolkow and
Baumann {vide p. 606).

Although thought to be especially frequent in various forms of tuberculosis,
alcajDtonuria must not be looked upon as specifically associated with any parti-
cular diseased conditions ; it indicates rather some peculiar independent changes
of metabohsm, and is not infrequently met with in conditions of apparent
health. In one case where there was a tendency for homogentisic acid to
appear in the urine, it was found that the quantity of this substance and the
associated colour phenomenon might be enormously increased by administering
tyrosine, and it is suggested that, when homogentisic acid or other aromatic
substances appear in excess, it is due to the action of special micro-organisms
on the tyrosine of the bowel.

Drug pigments. — The urine may contain purely accidental pigments due to
the use of drugs, notably of rhubarb, senna, logwood, and santonin.

The Inoeganic Constituents.

To a large extent the inorganic constituents of the urine arise
directly from the food, which always contains a large excess of salts.
It does not follow, however, that the bases and acids are to be found in
the urine in the same combinations as when ingested, and indeed an
interchange of base and acid may occur in special circumstances between
the salts of the food and those of the tissue fluids. Thus, excess of
potassium in the food may lead to increased elimination of sodium
(Bunge). The sulphates, moreover, form an exception to the general
rule of direct origin from the ingesta, very small quantities of these
salts being present in a normal dietary. The urinary sulphates are
derived almost entirely from proteid metabolism ; a small proportion of
the phosphates arising in the same way.

Sulphuric acid and other sulphur compounds. — About 80 per
cent, of the total sulphur in normal human urine is present in the
fully oxidised form of suljjhuric acid ; from 2 to 2-5 grms. of the acid.



6" ULPHURIC A CID AND S ULPHUR COMPO UNDS. 6 3 1

combined as salts, being excreted per diem. Two forms of salts exist —
(1) the ordinary and strictly inorganic sulphates of the form M'2S04,
and (2) the so-called conjugated or ethereal sulphates, which contam
organic radicles ; the composition of these will be clear from what
follows.

If ordinary alcohol be boiled with its own bulk of strong sulphuric
acid, under a vertical condenser, a crystalline product is formed which
is known as ethylhydrogen sulphate, or sulphovinic acid. The com-
position of this is explained by the following equation : —

C,H..OH H— 0. H.,0 CH— 0.

+ >S0o= " + ^ >so,

H— 0/ ^ H— 0— /

(alcohol) (sulphuric acid) (water) (ethylhydrogen sulphate)

By elimination of water, the ethyl radicle (C2H5 - ) becomes " con-
jugated " with the sulphuric acid, and a monovalent acid is formed,
which yields salts of the type M'CoH^ : SO4. On boiling such salts
with hydrochloric acid, the sulphovinic acid is first hberated, and then,
by absorption of water, splits up once more into alcohol and sulphuric
acid.

The " conjugated sulphates " of the urine are precisely analogous
salts, which undergo like decomposition when boiled with HCl. But,
instead of ethyl, the radicles conjugated with the sulphuric acid are
nearly always derived from aromatic precursors. In fact, as we have
already seen, most of the aromatic compounds of the urine described on
p. 605 e,t seq. are present as conjugated sulphates ; and the proportion of
the sulphuric acid present in this form depends upon the factors which
increase or decrease these aromatic substances. The chief salts present,
therefore, under ordinary circumstances, are those of kresyl- indoxyl-
and skatoxyl-sulphuric acids,

Normally, the sulphuric acid so combined amounts to about one-
tenth of the whole ; nine-tenths being in the form of ordinary sulphates.
An increased proportion of ethereal sulphates is found when, for any
reason, there is increase of proteid putrefaction in the body, and especi-
ally in the bowel; and also when larger amounts of aromatic compounds
than usual are taken by the mouth. In man they may be greatly in-
creased during starvation, whereas, according to I. Munk, they are absent
from the urine of a starving dog.

For the detection and estimation of the sulphates we rely upon the forma-
tion of the insoluble barium, salt. All the sulphuric acid originally present as
ordinary sulphates is precipitated as white crystalline barium sulphate, when a
soluble barium salt is added to the urine, previously made acid with acetic acid.
On the other hand, the barium salts of the conjugated sulphuric acids are soluble,
so that when the barium precipitate, obtained as above, is filtered off, the
ethereal sulphates still exist in the filtrate. But, as we have seen, they are
decomposed on boihng with hydrochloric acid, splitting up into sulphuric acid
and the hydrate of the conjugated radicle. It is evident, therefore, that if the
above-mentioned filtrate be so boiled with hydrochloric acid, a further precipi-
tate of barium sulphate may be obtained, the amount of which will be a
measure of the proportion of ethereal sulphates present.

One-fifth of the total sulphur of the urine is present, not in any form
of sulphate, but in less oxidised compounds. This fraction may be
spoken of as the " neutral sulphur" in contradistinction to the " acid



632 THE CHEMISTRY OF THE URINE.

sulphur " of the sulphates.^ We have but little knowledge of the actual
forms in which this neutral sulphur is excreted. As one source of the
imoxidised sulphur compounds, we may look to the taurin of the bile,
since it has been shown (in the dog) that when the bile is diverted
from the bowel by means of a fistula, the neutral sulphur of the urine is
diminished ; experimental or pathological blocking of the bile duct, on
the other hand, increases it.^ A second portion is probably present in
a compound or compounds analogous to cystine,^ and in actual cystinuria
the neutral sulphur is, of course, greatly increased (cf. p. 603).

Minute quantities of sulphocyanides are always present, probably
owing to reabsorption from the saliva which is swallowed, and these
contribute to the "neutral" sulphur.* But none of the sources we
have mentioned will account for the whole of the unoxidised sulphur
present, which must partly exist in compounds of which we have no
knowledge.

To estimate the neutral sulphur, a small quantity of the urine is evaporated,
and the residue fused with alkaline carbonates and nitrate of potassium. By
this means the whole of the sulphur present is oxidised to sulphates, and these
are estimated as barium sulphate. A separate estimation of the sulphuric acid
originally present is made, and the amount deducted from the figure obtained,
as above. ' The excess is a measure of the neutral sulphur, in terms of sulphuric
acid.

Phosphoric acid. — The greater part of the phosphates of the
nrine is derived directly from those ingested with the food, but a small
proportion arises from the oxidation of the nuclein, lecithin, and
protagon of the tissues. The phosphates are increased by animal food,
especially when this is rich in nucleo-proteids (Weintraud), and are
diminished by vegetable diet. The phosphoric acid of plants is mostly
present as insoluble earthy phosphates, which are not absorbed. On
this account the urine of herbivora is notably poor in phosphates. In
man the quantity is necessarily very variable, and ranges from 1 to
8 grms. of phosphoric acid in the urine of twenty-four hours ; it com-
monly amounts to about 3 "5 grms.

The nature of the salts present has been fully discussed in the
section devoted to the chemical reaction of the urine. Part of the
phosphoric acid is present in combination with lime and magnesia, but
a greater part is combined with the alkalies. Some importance has
been attached to a change in the relative proportion of the " earthy "
and " alkaline " phosphates in diseased conclitions, the estimation being
made by adding anmionia to the urine and so precipitating the former.
But the information so obtained may be misleading, as whatever the
form of calcium or magnesium salt originally present in the urine {e.g.
sulphates or chlorides), a precipitate produced hj ammonia would contam
these bases as phosphates, since an interchange of acids would take
place with the alkaline phosphates. From alkaline urines magnesium
ammonium phosphate (triple-phosphate) frequently separates in char-
acteristic crystals ; and in the deposit from feebly acid specimens

' Salkowski, Virclwic's ArcJiiv, 1875, Bd. Iviii. S. 472.

2 Cf. Kunke], Arch. f. d. ges. Physiol., Bonn, 1877, Bd. xiv. S. 344.

^ Goldmann and Baumann, Ztschr. f. phvsiol. Chem., Strassbnrg, 1888, Bd. xii.
S. 254.

* Learcd, Proc. Boy. Soc. London, 1870, vol. xvi. p. 18 ; I. Miink, Vircliovfs Arcliiv,
1877, Bd. Ixix. S. 354.



HYDROCHLORIC ACID.



633



calcium phosphate is found in star-shajjed masses of fine prisms (stellar
phosphate) (Fig. 58).

Pathologically, a diminution of the urinary phosphates is seen in nephritis
(Purdy), and an increase is said to occur in certain nervous diseases. The
phosphates may he greatly increased in diahetes insipidus.

For the estimation of phosphoric acid the urine is first treated with acetic
acid and sodium acetate, and is then titrated with a standard solution of
uranium nitrate. Perrocyanide of potassium or cochineal tincture may he used
as an indicator to mark the end point of the titration.

Hydrochloric acid. — There can be little douljt that the greater
part of the hydrochloric acid of mine exists as sodium chloride, and it




Fig. 58. — A. Stellar phosphates ; B. Triple phosphates.

certainly arises mainly from the common salt present in the food. The
tissues and fluids of the body maintain a very constant content of sodium
chloride, any excess is at once excreted, and any diminution in the
supply immediately reduces the excretion. The amount in the urine
depends, therefore, in normal circumstances, almost entirely upon the
quantity ingested, and falls to a minimum during starvation, or when
a salt-free diet is taken. Pathologically, striking alterations in the
chlorides of the urine may be observed. Thus, whenever considerable
exudations occur, as in pneumonic processes, or where pleuritic effusion
is taking place, the consequent removal of chlorides from the blood may
lead almost to a cessation of their excretion ; and conversely, during the
reabsorption of such exudations, the urinary chlorides may considerably
increase, even when but little salt is being taken by the mouth. Apart
from such exudations, fever appears to have a specific effect in pro-



634 THE CHEMISTR Y OF THE URINE.

moting a retention of chlorides ; a fact for which we have no sufficient
explanation.^

Upon ordinary diet, about from 6 to 10 grms. HCl is excreted per
diem by a healthy adult.

To demonstrate the presence of chlorides, the urine is diluted, made
acid with nitric acid, and mixed with nitrate of silver solution ; a white
curdy precipitate of silver chloride falls, which, if filtered off, is found
to be soluble in ammonia.

To estimate the hydrochloric acid, a known quantity of silver salt is added,
together with nitric acid, to a measured amount of urine, taking care that the
silver is in excess. The precipitate is filtered off, and the excess of silver
titrated in the filtrate with a standard solution of ammonium thiocyanate,
ferrous sulphate being used as an indicator. Knowing the amonnt of silver
added, and that left in the filtrate, the diff'erence indicates that combined as a
chloride, from which the hydrochloric acid can be calculated (Volhard's
method).

Carbonic acid is found even in acid urines ; some 50 c.c. being
present per litre.^ In acid urines the greater part is not in firm chemical
combination, as it is driven out of solution by the passage of a stream
of air. But when the urine contains abundant fixed bases, and especially
when it is actually alkaline from these, considerable quantities of car-
bonates may be present, the urine of herbivora being exceptionally rich
in these.

Nitric and nitrous acids may be present in normal urine in the
form of salts, but in quite unimportant quantity.

The nitrates are derived, not from metabolism, but directly from the
food ; the nitrites are not present when the urine is first passed, but
appear to arise always from the nitrates, as an effect of the reducing
action of micro-organisms.

Silicic and hydrofluoric acids may appear in traces, simply as
an effect of the presence of their salts in various foodstuffs.

Sodium and potassium. — Of sodium about 5 grms. per diem is
excreted upon a mixed diet, and of potassium about half this quantity.
The proportion of the latter metal is increased when the dietary is more
exclusively composed of flesh, and it is raised during starvation, and in
febrile conditions.^ We have already referred to the interesting fact
that the ingestion of large quantities of potassium salts may lead to
increased elimination of sodium from the body, and it is this driving out



Online LibraryE. A. (Edward Albert) Sharpey-SchäferText-book of physiology; (Volume v.1) → online text (page 89 of 147)