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

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of silver solution, be decomposed by sulphuretted hydrogen, and the filtrate
from the silver sulphide acidified with hydrochloric acid, concentrated, and
allowed to stand, the uric acid crystallises out. This being filtered off", the
liquid is again made alkaline with ammonia and the bases again precipitated
with silver nitrate. The varying solubility of the silver compounds so
obtained in nitric acid permits of a preliminary fractionation of the bases ;
and, when liberated from combination with silver, their diverse solubilities in
water and other media yield methods for their final separation from each

If urine (100 c.c.) be heated to boiling, and precipitated with a mixed
solution of copper sulphate and sodium thiosulphate, some chloride of barium
being afterwards added, a precipitate is obtained which contains all the uric
acid and xanthin bases, but no other nitrogenous constituent (Kriiger and
Wulff). By estimating the nitrogen in this precipitate by means of Kjeldahl's
process, we obtain a measure of what may be called the " alloxuric nitrogen,"
an important urinary constant. If a separate estimation of the uric acid be
made, the nitrogen proper to this may be deducted from the "alloxuric
nitrogen," and we obtain a value for the "nitrogen of the bases." Such a

1 Kriiger and Wulff, Ztschr. f. jyjiysiol. Chem., Strassbiirg, 1896, Bd. xx. S. 176.
rNH— 00^

M I I .

"Alloxan-! CO CO r is an oxidation product of uric acid. The xanthin bases and uric

Inh— C0>'

acid will all be seen to contain the urea residue and the three-carbon chain, which together
comprise the so-called " alloxan ring."


procedure is our most convenient method for following the variations in the
excretion of the xanthin group. ^

Tests. — Xanthin and its two homologues, and also carnin, but not the
other bases, give Weidel's reaction. This is almost identical with the
murexide test described for uric acid, but chlorine water is used instead
of nitric acid. The bases resist oxidation with nitric acid much more
fully than does uric acid, but, in the presence of a small quantity of a
chloride, xanthin will give the ordinary murexide reaction. Very
characteristic of xanthin and hypoxanthin are the crystalline precipi-
tates which they yield with silver nitrate in the presence of nitric

Variations in the amount of the urinary xanthin bases closely follow
those of uric acid, and for the most part depend upon the same
influences. The bases, however, are apt to vary even more widely.
According to Camerer ^ they are greatly increased by certain forms of
vegetable food ; thus, in one experiment, on a flesh diet the nitrogen
present as these bases was only O'l per cent, of the total nitrogen ;
while, when green vegetables formed the chief ingredient of the food, it
was 0-6 per cent. They are increased by diet rich in nucleins,^ and
pathologically their amount is greatly raised in some forms of leukaemia.

(f) Creatinin.— This base is chemically distinct from the alloxuric
compounds, in that its molecule contains neither the alloxan ring nor
the urea residues which are characteristic of these. Nevertheless, on
hydrolysis, it easily yields urea and an amido-acid (methylglycine). It
is the anhydride of creatin, which is itself methylglycocyamin.

/NH2 /NH

C(NH)/ C(NH)/ "-^^^

\n(CH3)-CH-C00H \N(CH3)-CH-C0

(creatin) (creatinin)

Whether creatin itself is ever a urinary constituent is somewhat
uncertain. It has been stated to occur when the urine is excreted in
alkaline condition,* but the quantity is, in any case, very small. So
easily are the two substances converted the one into the other, that care
is requisite in the isolation of either. When creatin stands in acid solu-
tion, it tends to change into its anhydride, while creatinin in alkaline
solution suffers the inverse change.

Gr. S. Johnson^ has found, however, that urinary creatinin is not
identical, but isomeric, with that obtained artificially from creatin (e.g.
by the action of acids), and distinct from the creatinin found in small
quantity in muscles.

The creatinin of urine was first isolated by Liebig. It is present on
an average to the extent of about I'O grm. in the excretion of twenty-
four hours, when a mixed diet is taken.

ProiJerties. — In its compounds creatinin exhibits well-marked basic
tendencies, and it can liberate ammonia from ammonium salts on boil-
ing ; but, according to Salkowski, solutions of the pure substance react

1 For details see Krliger and Wulff, loc. cit. supra.

- Ztschr.f. Biol., Miinchen, 1891, Bd. xxviii. S. 72.

•* Weintrand, loc. cit.

•• Hofremaiin, Vircliovfs ArcMi\ 1869, Bd. xlviii. S. 358.

^ Froc. Hoy. Soc. London, 1892, vol 1. p. 287.


neutral to litmus. The crystalline form of the base varies with the
method of preparation (Johnson). As ordinarily obtained, it exists as
colourless monoclinic prisms, which are often imperfectly formed, and
appear of whetstone shape (Fig. 53). It dissolves in about twelve parts
of cold water, but requires a hundred parts of alcohol to dissolve it at
ordinary temperatures. In ether it is almost insoluble. Creatinin
reduces alkaline copper solutions (cf. p. 608). It forms characteristic
crystalline salts with the mineral acids, aqueous solutions of which react
acid to litmus. With certain salts of the heavy metals it forms crystal-
line molecular compounds, two of which are of practical importance.

Creatinin zinc-chloride — (C4H7]S[30)2ZnCl2 — separates as a precipi-
tate, consisting of stellate clusters of acicular crystals, when a concen-
trated neutral solution of chloride of zinc is added to an aqueous or
alcoholic solution of the base. The compound is soluble in hot water,
in mineral acids, and in alkalies ; but insoluble in alcohol, and very
slightly soluble in cold water.

Creatinin niercnric-cliloride, a complex compound of the formula
4(C4H7]Sr30.HCl.HgO),3HgCl2. This is precipitated in colourless, glassy,
spherular masses, when sodium acetate and mercuric chloride are added to
creatinin solutions. The base is also precipitated, even from very dilute
solutions, by the addition of phosphotungstic, phosphomolybdic, or picric

Isolation and estimation. — JSTeubauer separated creatinin frora the urine by
means of its combination Avith zinc chloride, this salt being added to an
alcoholic extract of the evaporated urine. A more convenient method is to
treat the urine direct with a little sodium acetate, and then with one-fourth
its volume of saturated mercuric-chloride solution. The precipitate which
first falls is at once filtered off; it contains uric acid and other constituents,
but not creatinin. The filtrate from this rapidly begins to deposit the mer-
cury compound described above, and in forty-eight hours precipitation is com-
plete (Gr. S. Johnson). The base itself is prepared by decomposing this
precipitate with sulphuretted hydrogen, and by treating the creatinin-hydro-
chloride, so obtained, with hydrate of lead.^ To determine the quantity, the
mercury precipitate may itself he weighed, and the percentage of creatinin
calculated from this.^

Tests. — If a solution of creatinin be treated with a small quantity of
very dilute sodium nitroprusside solution, and subsequently with weak
caustic alkali, a rich, ruby-red colour is produced, which afterwards
changes to yellow {Weyl's reaction). If acetic acid be now added in
excess, and heat applied, the solution becomes green, and then blue,
and finally a precipitate of Prussian blue is formed. Acetone (p. 616)
gives an analogous reaction, but behaves differently after the addition of
the acetic acid. Many specimens of urine will give Weyl's test direct.

Jaffa's test is an application of the fact that creatinin gives, with
picric acid and caustic alkali, an intense red colour, even in the cold.

The variations in the urinary creatinin generally follow very closely
those of the urea, but there can be no doubt that its quantity depends
largely on the amount of creatin taken with the food. Its physiological
relations are discussed elsewhere. Pathologically, it is increased in most
febrile conditions, and in diabetes. It has been stated to diminish in

^ In tliis process all the operatious are carried out in the cold ; by this means the true
urinary creatinin is obtained. Heat produces isomeric change.
- Cf., however, Allen, "Chemistry of Urine," pp. 156 and 159.



progressive muscular atrophy, and in pseudo-hypertrophic paralysis.
According to Senator, no increase is produced by the paroxysms of
tetanus — a fact which is of interest as bearing on the relation of
muscular activity to the urinary creatinin.

(g) Hippuric acid. — Hippuric acid is benzamido -acetic acid, or
benzoylglycin, C6H5.CO.NH.CH2.COOH ; in other words, it is a con-
densation product of benzoic and amido-acetic acids, in the formation
of which the hydroxyl group of the former is eliminated as water,
with an atom of hydrogen from the amido group of the latter. But

Fig. .53. — A. Creatinin ; B. Hippuric acid.

the simplest artificial synthesis is obtained when monochlor-acetic acid
is heated with benzamide.


In most mammals the synthesis by dehydrolysis occurs in the kidney ;
hippuric acid appearing in the urine, whenever benzoic acid, or pre-
cursors of benzoic acid, are taken by the mouth. The excretion of
hippuric acid is, indeed, mainly dependent upon the relative richness of
the diet in such precursors of benzoic acid.

It is not necessary that benzoic acid should itself be ingested. A
benzene derivative containing a single "side-chain" is nearly always
oxidised in the body to benzoic acid. Such substances, therefore, as
toluene, Cf;H5.CH3 ; cinnamic acid, CeH5.CH.OH.COOH ; or phenyl-
propionic acid, CgHs.CHg.CHa.COOH, all give rise to an excretion of
hippuric acid when they are taken by the mouth. Aromatic compounds
of this type are abundantly present in some forms of vegetable food, as


in many fruits and in the cortical parts of most jjlants. Vegetable food
greatly increases, therefore, the excretion of hippuric acid.

But the vegetable aromatic compounds are not the sole source of the
urinary hippuric acid. In the decomposition of proteids, which occurs
in the bowel, aromatic residues split off. Precursors of benzoic acid
(mainly, perhaps, phenylpropionic acid) are thus formed, and after
oxidation they appear in the urine as hippuric acid. The metabolism
of the tissue proteids themselves, moreover, may yield precursors of the
same kind, so that even in starvation hippuric acid does not wholly dis-
appear from the urine.

This dual origin (from aromatic precursors in the diet chiefly, but
likewise from proteid metabolism) is found also in the case of the other
aromatic constituents of the urine (p. 605).

Upon a ] nixed diet the excretion of hippuric acid hi human urine
amounts to about 0'7 grms. per diem ; upon a diet rich in fruits it may
be raised to three or four times this. In herbivora the quantity is much
larger; the urine of cattle, for instance, often contains as much as
2 per cent., though, as might be expected, that of sucking calves only
contains small amounts.

Properties. — It forms four-sided prismatic crystals ending in two or
four facets, and often grouped in clumps (Fig. 53), of which the melting
point is about 187°. It is but slightly soluble in cold water or alcohol ;
but both these solvents dissolve it easily when hot. It is soluble in
acetic ether, but not so in most other organic liquids. If heated to 240°
it decomposes, benzoic acid subliming out and a reddish residue being left
behind. When first heated at this temperature a hay-like odour is given
off, which is succeeded by that of prussic acid. When boiled with strong
hydrochloric acid it splits up into its components, benzoic and amido-
acetic acids. The growth of the Micrococcus urem can bring about this
decomposition, so that stale specimens of urine often contain benzoic in
place of hippuric acid. Taken to dryness with nitric acid, it yields an
odour of nitrobenzene.

Solutions of hippuric acid react acid to litmus, and even when very
dilute they impart a violet colour to congo-red. By the use of the latter
indicator Briicke proved the absence of the free acid from the urine. It
is present always as salts. It forms salts with bases, but does not
combine with acids. Its iron compound is insoluble in hot water, and
may be employed in separating the acid from its solutions.

Isolation and estimation. — The method of Bunge and Schniiedeberg ^
consists in making an alcohoHc extract of the urinary soHds, evaporating off
the spirit, dissolving the residue in water, and, after acidifying with hydro-
chloric acid, shaking up repeatedly with successive quantities of acetic-ether.
On evaporating the latter, impure crystals of the acid are obtained, the
impurities being removed by treatment with petroleum-ether, in which
hippuric acid is insoluble.

Tests. — The substance is recognised by its crystalline form, by its
melting point, by its behaviour on heating, and by the formation of its
insoluble iron compound when neutral ferric chloride is added to its

In addition to hippuric acid, minute quantities of its homologue
phenaceturic acid (phenylacetylglycin), CeH^.CHoCO — NH.CHo.COOH,

■^ Arch. f. expei: Path. it. Pharmakol., Leipzig, Bd. vi. S. 235.



are occasionally found in human urine. Its origin and significance are
analogous to those of the more abmidant substance.

When benzoic acid or its precm-sors are administered to birds, they
are excreted as ornithuric acid, which is an analogous conjugated com-
pound of benzoic acid with diamidovalerianic acid.

(h) Araido- acids. — These, in simple unconjugated form, are seldom
found in normal urine. Under certain pathological conditions leucine
and tyrosine appear in considerable quantities. The elimination of these
substances is especially associated with conditions in which a rapid
destruction of the hepatic tissue has occurred ; thus they are found in
acute yellow atrophy of the liver, and, to a less extent, in phosphorus-

When these amido-acids are given by the mouth in moderate
quantity, and under conditions of normal health, their nitrogen is
excreted wholly in the form of urea. If, however, tyrosine be adminis-
tered in very large amounts, it may be excreted in part as tyrosine-
hydantoin, in which it exists as a conjugate compound with urea ; ^ and
at the same time other aromatic constituents of the urine are increased
in quantity by derivation from its aromatic nucleus. Only when the
normal hepatic functions are in abeyance does the unaltered aniido-acid
itself appear.2

When present in urine, leucine and tyrosine are usually found
together. If in large quantity, they may, though very rarely, form
a deposit ; at other times they may be seen under the microscope
when a drop of the urine is evaporated.

In general, however, they must be
separated by special means. The
leucine may be dissolved, by means of
hot alcohol, from the residue obtained
by evaporating the urine, and when
the alcoholic extract cools it separates
as a greasy mass, which under the
microscope will be seen to consist of
minute spheroids with concentric mark-
ings interrupting a radiated structure.
To demonstrate the presence of tyrosine,
the urine is first precipitated with
basic acetate of lead, the fihrate from
the lead precipitate treated with sul-
phuretted hydrogen and again filtered.
On thorough concentration and cool-
ing of the lead-free fihrate, the tyrosine

separates out in characteristic acicular prisms, which are mostly combined

into sheaves or stars (Fig. 54).

Leucine and Tyrosine.

Cystine ^ is another amido-acid, but it is at the same time a sulphur-
containing substance, differing in its metabolic significance from leucine
and tyrosine.

^ Jaffc5, Zlsehr. f. 'jyhysiol. Cliem., Strasslmrg, 1883, Bd. vii. S. 306.

2 According to tlie recent observations of Ulrich, leucine and tyrosine are always to be
found in normal urine, though in small quantity, Ccntralbl. f. PhiisioL, Leipzig u. "Wien,
]897, Bd. xi. S. 12. J J > I ^

" Of. Baumann, Ztschr. f. pliysiol. Chem., Strassburg, 1884, Bd. viii. S. 299 ; also
Bren.singer, ihid., 1892, Bd. xvi. S. 5.o2.



It is a sulphur derivative of an amidolactic-acid, and has the
formula :

CI13 C/II3

1 I

NH2— C— S— S— C— NK,


It may appear in small quantity in certain diseases, Ijut is generally
a product of peculiar disordered metabolism, which is found to be char-
acteristic of certain families. Members of such families may excrete
habitually from 0'5 to 1 grm. daily. It sometimes separates as a crystal-
line deposit from the urine, and occasionally forms calculi in the urinary

Physiologically it is of interest, in that cystine or substances allied to
it are probably the precursors of certain of the normal sulphur compounds
of the urine (]3. 632).^

Its crystals are very characteristic, being usually in the form of
hexagonal plates (Fig. 55) ; more rarely it appears in rhombohedral
form. Urine which contains it will, if heated with caustic potash and
plumbic acetate, give a black precipitate of lead sulphide.


Normal urine contains but traces of substances belong-ino; or allied
to the proteid group. But minute quantities of a nudeo-2)roteid derived
from the cells of the urinary passages
are seldom or never absent. In the
majority of cases the amount of this
is so small that it is difficult directly
to demonstrate its presence. The
flocculent cloud which generally
separates on standing, even from the
clearest urine, by no means always
contains any isolated proteid, but may
consist entirely of intact epithelium
cells. But the nucleo-proteid may
be detected by suitable tests in the
precipitate which falls when large
quantities of normal urine are mixed
with alcohol.

The nucleo-proteid may, on the
other hand, so far increase in con-
ditions of apparent health, that the urine will react to Heller's test
{vide infra). Thus Plensburg ^ found, on examining the urine of 1252
healthy persons, that 97 of these gave a reaction with nitric acid, which
could be shown to be due to a nucleo-proteid.

In such cases, and in others where the increase is greater and due to
inflammatory changes in the urinary tract, the nucleo-proteid may be
precipitated by the addition to the urine of acetic acid in the cold ;
especially if the fluid be first diluted to eliminate the solvent action of

^ Goldmann and Baumann, ibid., 1888, Bd. xii. S. 254.
^ Skandin. Arch. f. Physiol., Leipzig, 1893, Bd. iv. S. 410.

Fig. 55. — Cystine.


the salts present, or, better still, if the salts be first reduced by dialysis.
In some pathological conditions, and especially in cystitis, the amount may
be so greatly increased that it separates as a viscid gelatinous precipitate.
The mucoid appearance of the urinary nucleo-proteid led to its being long
looked upon as mucin ; but it does not yield a reducing substance on
hydrolysis, while, on the other hand, it is rich in phosphorus. Nevertheless,
recent researches made upon large quantities of urine indicate that the
precipitate given by acetic acid contains small quantities of ordinary
mucin, or a phosphorus-free mucoid, as well as the nucleo-proteid.^

Apart from increase due to inflammatory conditions of the excretory
tract, nucleo-proteid is said to be increased when the blood is excep-
tionally rich in leucocytes (leuka'mia).^

The question as to whether or not normal urine contains serum albumin
or serum globulin offers a problem of the same order as that of physio-
logical glycosuria, fully discussed on p. 608. The matter is, however, of
less importance physiologically than is the latter question, as, although the
evidence to hand points to the fact that if sufficient urine is employed these
proteids may nearly always be separated in minimal traces, it by no means
follows that they form part of the true excretion, for they may arise
rather, like the nucleo-albumin, from the surface of the urinary tract.

As to the cases when, in apparent health, there is such an increase of
these proteids that their presence may be shown by the direct applica-
tion of ordinary tests, we are met with the difficulty of having to define
what is meant by "normal" urine. Such quantities may be present,
for instance, after exceptionally severe exercise, as in the urine of soldiers
after prolonged marching (Leube, Chateauburg) ; but it is not certain
that the excretory mechanism is here working physiologically.

When, as the effect of disease, the renal epithelium has undergone degenerat-
ive changes, the presence of albumin in the urine is a common phenomenon ;
one of the most familiar in pathology. Albuminuria may arise, too, from such
alterations in the constitution of the blood as upset its normal relations to the
renal cells ; this may be observed in anaemia, and as the effect of specific
poisons. Again, it may follow disturbances of blood pressure in the renal
vessels, even though these be unassociated with obvious changes in the
excretory epithelium. Lastly, the albumin due to addition from the excretory
tract, after the urine has left the kidneys, may pathologically reach a consider-
able proportion.

Under pathological conditions, also, the urine may come to contain
albumoses and peptones. On the one hand, a so-called enterogenous peptonuria
or albumosuria may occur, when, from degenerative changes in the gastro-
intestinal walls {e.g. in carcinoma ventriculi or the ulcerative stage of enterica),
the diffusible proteids reach the blood stream and thereupon are immediately
eliminated by the kidneys. On the other hand, these substances may reach
the blood stream from abscesses or other purulent collections where the tissue
proteids have been hydrolysed by the growth of organisms. Whatever their
origin (and it is sometimes not so clear as in the above groups of cases), the
proteoses and peptones no sooner reach the blood than they are found in the
urine. The older methods of investigation did not clearly distinguish between
peptones and albumoses in the urine; evidence is now accumulating to show
that the latter are by far the more common.

1 Cf. Malfatti, Wien. Iclin. Wchnschr., 1891, S. 433; also Mcirner, Skandin. Arch,
f. Physiol., Leipzig, 1895, S. 437.

- According to recent observations, the nucleo-proteid of nrine is in some cases to be
identified with Lilicnfeld's "nucleohiston."


A very large number of tests for the presence of albumin and globulin in
the urine have been described. We can here refer to two only.

Heller^s test. — A small quantity of strong nitric acid is placed in a test
tube, and the urine is allowed to flow gently down the side of the tube so that
it floats upon the surface of the acid without mixing with it. If coagulable
proteids are present, a dense white ring forms at the junction of the liquids.
As little as '002 per cent, of albumin may be thus detected. The urinary
nucleo-albumin may react to this test if in sufficient quantity, but the ring
formed is less dense, and more apt to be formed at some little distance from the

Feri'ocyanide test. — A solution of potassic ferrocyanide is first added to the
urine, and the mixture made acid with acetic acid, when the albumin and
globulin are precipitated as a flocculent cloud. If the salt be added before the
acid, nucleo-proteid is not precipitated.

To separate serum globulin from albumin, the urine is, after neutralisation,
saturated with magnesium sulphate, which precipitates the former. The
precipitate may contain certain of the salts of the urine, and heteroalbumose
if present. The proportion of globulin to albumin may vary greatly, and may
be quite different from that present in the blood.

To detect peptones, the urine is saturated with sulphate of ammonium, and,
after standing, filtered ; the biuret test may now be applied to the filtrate, a
large excess of caustic alkali being used. The ammonium sulphate precipitate
contains (in addition to ammonium urate) all other proteids present and also
the urinary mucin. If this precipitate be allowed to stand under alcohol for
some days, the proteoses are obtained in solution when it is extracted with

Online LibraryE. A. (Edward Albert) Sharpey-SchäferText-book of physiology; (Volume v.1) → online text (page 85 of 147)