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

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A crystalline combination of urea with phosphoric acid is also known, and
others with various organic and inorganic acids.

Crystalline compounds are also formed with certain neutral salts ; that
with sodium chloride is said occasionally to form when urine is concentrated
on the water bath. The compound with palladium chloride is very insoluble.
A molecular combination with hasic mercury niti-ate is quite insoluble in water,
and is of historic interest, as its formation is the basis of the classical method
of urea estimation suggested by Liebig in 1853 {vide infra).

When fused and gently heated after fusion, urea yields biuret and
cyanuric acid. Two reactions occur as follows : —

2NH2.CO.NH2 = NH2.CO.NH.CO.NH2 + H3N ; and


3NH2.CO.NH2 = C3N3(OH)3 + 3(H3N)

(cyanuric acid)



Its relations to ammonium carbonate and carbamate are very im-
portant from a physiological standpoint.


The two molecules of water necessary to form carbonate of ammonia
are very readily taken up. Even at a temperature of 60° C, an aqueous
solution of urea slowly develops ammonia (Leube ^) ; while a boiling
solution decomposes with considerable rapidity. Heated with water


= C0< 4-H,0


= co<

+ H,0


(ammonium carbamate)

(ammonium carbonate)

Fig. 49. — Upper half, urea nitrate crystals,

Lower half, urea oxalate

under pressure at 180°, the conversion into ammonium carbonate is
quickly complete. A solution of pure urea may be evaporated at tem-
peratures from 60° to 75°, without serious loss, but in the urine it is less
stable. Quite appreciable proportions of its nitrogen are lost as ammonia
when itrine is evaporated, even at low temperatures. In the presence
of free acids and bases, the hydrolysis occurs with still greater readiness,
the ammonium carbonate formed being further decomposed by the
reagent. Thus, on boiling urea solutions with acids, carbonic acid is
given off"; on boiling them with alkalies, free ammonia is evolved.

Tlie hydrolysis is also induced by micro-organisms, as in the
ammoniacal fermentation of urine. The Micrococcus urcm is the best
known of these ; but other organisms are found in decomposing urine

^ Firchow's Archiv, 1885, Bd. c. S. 552.

UREA. 583

which can produce the same result. On the other hand, urine may
develop many organisms which have no such power.^ So long as the
bacteria which induce the change are alive, the enzyme is closely
associated with the living cell, and a filtered urine is ferment free
(Sheridan Lea). But when the cells are dead, a ferment may be
extracted from them which hydrolyses pure urea solutions.

While urea is thus easily converted into ammonium carbonate, the
intermediate substance ammonium carbamate (formed by the action of
dry CO2 upon NH3), if heated to 135°, or treated with alternating electric
currents, splits up into urea and water. The hepatic cells have the
power of dehydrolising ammonium carbonate itself to form urea. Nitrous
acid and the hypobromites oxidise urea according to the following
equations : —

(1) CO(NH2>,+2¥OOH=C02+2N2+3H,0

(2) CO(NH2)o+3BrONa=3NaBr+CO,+N,+2H20

Separation of urea. — To prepare pure urea from urine, advantage may
be taken of the insolubility of the nitrate. The urine is concentrated to a
small bulk, and pure nitric acid is added in excess ; the mixture being kept
thoroughly cool during the addition of acid. The crystals are strained off by
pouring through muslin, and freed from excess of acid by pressing between
thick filtering paper. They are mixed with excess of barium carbonate,
sufficient alcohol is added to form a paste, and the mixture dried on the water
bath. On extracting the dried residue with absolute alcohol, a fairly pure
solution of urea is obtained, from which crystals separate on evaporation.

I find that fine crystals may be prepared by the following simpler method.
Half a litre of urine is evaporated to a thick syrupy consistence, and the
residue is exhausted with hot absolute alcohol. The spirit is filtered and
taken to dryness ; and the residue extracted on the water bath with successive
quantities of pure acetone, which should be filtered while hot. The mixed
acetone extracts are evaporated nearly, but not quite, to dryness. On cooling,
fine white crystals of urea separate, any pigment present remaining in solution
in the small quantity of the solvent which is allowed to remain. The crystals
may be washed with cold acetone.

Tests. — For the detection of urea, the formation of the characteristic
crystals of the nitrate or oxalate (and especially of the former) is of
practical value. On the small scale the process of crystallisation may
be watched under the microscope ; a drop of the suspected fluid, after
concentration if necessary, and another of nitric acid, being allowed to
run together on a glass slide.

The formation of biuret is an excellent test for urea, if the crystals
are first obtained in moderate quantity and fairly pure. After heating
them, as described above, the residue is dissolved in water, excess of
caustic alkali is added, and one or two drops of a dilute copper sulphate
solution. A pink colour is produced like that given by peptones under
like circumstances (" biuret reaction ").

Estimation of urea. — K'o method is known by which urea can be separated,
as such, from the urine in a quantitative manner. The ease with which it is
hydrolised is a fundamental difficulty in the way of such quantitative isolation.
We can, however, find precipitants for the other nitrogenous constituents, and
a determination of the remaining nitrogen after the removal of these gives the
best available measure of the urea.

^ For information on this subject, vide Leube and Graser, Virchows Archiv, loc. cit.\ and
Warrington, Jonm. Cliem. Soc. London, 1888, vol. i. p. 727.


The most satisfactory of the methods based upon this principle is that of
Morner and Sjbquist.^ To carry out this process, 5 c.c. of the urine is treated
with an equal volume of a saturated solution of barium chloride containing
5 per cent, of caustic baryta ; 100 c.c. of an alcohol-ether mixture (2-1) is
added, and the whole allowed to stand for twenty-four hours in a closed flask.
After filtering from the precipitate the solution is evaporated at low tem-
peratures (below 60°), and a determination of nitrogen made, by Kjeldahl's
method, in the residue. By the precipitation thus described all nitrogenous
substances are removed except urea and ammonia, while the last is got rid of
during the evaporation of the filtrate. The percentage of nitrogen found
multiplied by 2"143 will give the percentage of urea.

When less accuracy is required, the well-known process of Knop- and
Hiifner is now universally employed. This depends on the decomposition of
urea by the action of hypobromites ; the nitrogen Avhich is evolved being
measured in a graduated tube, and the urea calculated from the amount thus
found. The equation for this reaction is given above (p. 583). The solution
of sodium hypobromite employed contains excess of caustic alkali, so that
the carbon dioxide which is formed simultaneously with the free nitrogen,
is retained in solution as carbonate of sodium. Only some 92 or 93 per
cent, of the total nitrogen present as urea is obtained in this process, the
remainder being converted into cyanates. On the other hand, the uric acid,
creatinin, and other nitrogenous substances present yield a proportion of their
nitrogen, so that part of this error is counterbalanced. Many varying in-
fluences affect the result, however ; diabetic urine, for instance, is said to
yield a greater proportion of its total nitrogen, owing to the effect of the
sugar present. It should, in fact, be clearly understood that the hypobromite
process, while of great convenience and of sufficient accuracy for clinical and
many other purposes, does not give a scientific measure of the urea. The
calculation of its results is best made by taking each 37'1 c.c. of nitrogen
measured at ordinary temperatures as equivalent to one decigramme of urea.^

The titration method of Liebig referred to on p. 581 is now of little more
than historical importance, though it was used in all the older work upon
metabolism. It depended in principle on the fact that urea, under carefully
defined conditions, forms a definite insoluble compound with basic mercuric
nitrate. A standard solution of nitrate of mercury was added to the urine
until the Avhole of the urea was precipitated in this form, the end-point being
marked when a drop of the urine gave a yellow colour Avith sodium carbonate
(indicating excess of mercury). The modifications necessary for accuracy have
been carefully worked out by Pfliiger and others ; in its perfected form, however,
the jjrocess becomes one for the estimation of the total nitrogen of the urine
rather than for the urea only, and for this purpose it is entirely superseded by
Kjeldahl's method {supra, p. 580).

The variations in the quantity of urea present in the lu'ine are
dealt with in the article on metabolism, where their cause is dis-
cussed. The average quantity excreted by a healthy adult man under
normal circumstances is about 30 grms. per diem ; that is to say, the
m'ine will contain about 2 per cent. Its absolute amount is necessarily
increased by all causes which stimulate nitrogenous metabolism, but the
iwoportion which the urea bears to the other nitrogenous constituents is
an independent variable {vide infra).

^ Skandin. Arch. f. Physiol., Leipzig. 1891, Bd. ii. S. 438; Jahresh. u. d. Fortschr.
d. Thier-Chem., Wiesbaden, Bd. xxi. S. 168; cf. also Bridtker, Ztschr. f. 2')liysiol. Chem.,
Strassbiu-g, 1893, Bd. xvii. S. 146.

^ The original description by Knop will be found in Chem. Ce.nlr.-Bl., Leipzig, 1860,
S. 244. Details of various modern modifications are found in most iiractical handbooks.

» Cf. A. H. Allen, "Chemistry of Urine,"' p. 148.


According to Tschlenofi',^ if the urea excretion after a meal rich in
proteicls be estimated from hour to hour, it will be found to exhibit two
maxima. The first occurs at the third or fourth hours, and the second at
the sixth or seventh. These he considers to indicate the absorption of
peptones from the stomach and intestine respectively. If peptones be^
given instead of ordinary proteids, the maximum is reached l^y tlie second
hour. Mares,^ on the other hand, found that after an isolated meal the
maximum of urea excretion was not reached till the ninth hour.
Kobler ^ has found that simple diuresis under normal circumstances is
not accompanied by increased excretion of urea.

(c) Aniinonia. — The urine of man and of carnivorous animals
invariably contains small quantities of ammonium salts. They may
be absent, however, from that of lierbivora. The quantity in human
urine is about 0"7 gnn. NH3 per diem ; the variations in health extend-
ing from about 0"3 to 1"2 grms.^

The ingestion of annuonium carbonate, or of organic ammonium
compounds susceptible of oxidation in the body, does not increase the
excretion of ammonia, for the nitrogen of such compounds is excreted
wholly as urea. If, however, stable salts of ammonium, such as the
chloride, are given, they appear (in the case of carnivora, at any rate) as
such in the urine.

Apart from such direct ingestion of stable ammonium salts, the
excretion of ammonia depends almost entirely upon that question of
adjustment between acid production in metabolism and the supply of
bases in the food which was discussed in the section devoted to the
acidity of the urine {g.v?). Ammonia formation is the physiological
remedy for deficiency of bases.

When acid production is excessive (a condition especially seen in
certain forms of diabetes), or when mineral acids are given by the
mouth, the urinary ammonia increases at the expense of the urea.
When the bases are in excess, whether from the nature of the food or
from the administration of alkalies, the ammonia disappears, and a corre-
sponding amount of urea is excreted in its place. From this it follows
that little or no ammonia is found in the urine of herbivora ; and that,
in man, flesh food raises the quantity, and vegetable food diminishes it.^

From the abundance of bases in their food, it is very difficult, by any
means, to increase the urinary ammonia of herbivora. If, for example,
abundant ammonium cliloride be given to a rabbit, togetlier with a normal
supply of vegetable food, its lU'inary ammonia is but little increased.*^ By
double decomposition with sodium carbonate in the tissues, ammonium car-
bonate and sodium chloride are formed, and the former is excreted as urea.

It Avould seem that the organisation of the herbivora does not permit of
a sujiply of ammonia to neutralise acids when given in excess. Thus, most
herbivorous animals are said to be much more susceptible to poisoning by
acids than are the carnivora.

^Abstract in Centralhl. f. Physiol., Leipzig u. Wien, 1896; cf. also Veragutt, Journ.
Physiol., Cambridge and London, 1897, vol. xxi. p. 112.

^ Jahrcsh. il. d. Leistung. . . . d. ges. Med., Berlin, 1887, Bd. i. S. 145.

3 JFien. klin. Wchnsclir., 1891, Nos. 19, 20.

^ Neubauer, Journ. f. prakt. Chem., Leipzig, 1852, Bd. Ixiv. S. 177. These figures are
confirmed by numei'ous later observers.

^ Salkowski and Munk, Virclww's Archiv, 1877, Bd. Ixxi. S. 500 ; also Gnmlicli,
Ztschr. f. physiol. Chem., Strassburg, 1893, Bd. xvi. S. 19.

^ E. Salkowski, Ztschr. f. 2-)hysiol. Chem., Strassburg, 1877, Bd. i. S. 26.


To demonstrate the presence of the small quantities of ammonia in
human urine is not easy, owing to the ready production of the base
by hydrolysis of urea, which must, obviously, lead to error. We must
employ a method analogous to that used for its estimation.

Estimation of ammonia (ScMosing's method). — Twenty-five c.c. of urine are
placed in a basin with vertical sides, and about 20 c.c. of milk of lime are
added. A glass triangle is placed over the basin, and, upon it, another small
vessel containing 20 c.c. of one-fifth normal sulphuric acid. These stand
upon a glass slab, and are covered with a bell-shaped glass cover, fitting air-
tight on the slab. The ammonia is liberated by the lime, without any
decomposition of other nitrogenous constituents, and, in the course of three
days, the whole is absorbed by the sulphuric acid, the degree of neutralisation
being afterwards estimated by titration. If dilute hydrochloric acid be
used instead of sulphuric, it may, after the experiment, be evaporated to
dryness on the water bath, and the residue taken up with a small quantity of
water. Platinic chloride added to this solution will demonstrate the presence
of ammonia, by giving a yellow crystalline precipitate of ammonio-platinic

Pathologicahy, the urinary ammonia may be increased, not only after the
manner we have discussed, by abnormal acid production (as in diabetes and
fevers), but also by conditions which reduce the proper activity of tbe
hepatic cells, whereby the dehydrolysis of ammonium carbonate into urea is
less complete than normally.

(d) Uric acid. — Uric acid was first separated from human urine by
Scheele, in 1776. It is present in the urine of most mammals, though
from that of the dog and cat it has been shown to be frequently absent.
In man the daily output in the urine varies considerably (from 0'2 grm.
to I'-i grm.), the average amount being O^S grm.

Chemical constitution. — Eightly to appreciate the physiology no less
than the chemistry of uric acid, its close relationship to urea shoald be
clearly understood. It yields the latter easily by a combined process of
oxidation and hydrolysis. It belongs, ui fact, to the class of substances
known as diuriiides, in which the residue of two urea molecules are
united to a carbon - containing nucleus. In the case of uric acid this
nucleus contains a chain of three carbon atoms.

The constitutional formula first suggested by Medicus —

NH— C— NH\




has now received ample confirmation from the synthetic production of
the acid by Horbaczewski,^ and by Behrend and Eoosen.^

The ureides are, in general, produced by the condensation of hydroxy-
acids with urea. The hypothetical acid, which would yield uric acid
by such simple condensation, would be a trihydroxyacrylic acid ; but
this has never been prepared.

Lactic acid also contains a three-carbon chain in its molecule, and,
because of the important physiological relationships of this acid, it is of
special interest to find that m'ic acid can be synthesised by linking urea

1 Monutsh.f. Chem., Wien, 1887, Bd. viii. S. 201, 584.
- Ber. d. deutsch. chem. Gesellsch., 1888, Bd. xxi. S. 999.



residues on to a nucleus derived from lactic acid. This Horljaczewski
succeeded in doing by heating urea with trichorlactamide : —

CC1.3CH.OH.CO.NH,+2(NH,3),CO=C5H,N,03 + NH.Cl + 2HCH-H,0

The simple changes involved in this reaction will be more clearly seen on
examination of the following graphic scheme : —













The groups printed in thick type unite to form uric acid ; the atoms repre-
sented in thinner type split off to form respectively a molecule of ammonium
chloride, two molecules of hydrochloric acid, and one of water.

Uric acid is formed also when glycine is heated with urea (Hor-
baczewski), but the molecular changes involved are not so simple
as those shown above, and the yield is not so good. In Behrend arid
Eoosen's synthesis the nucleus is primarily derived from acet-acetic
ether, and the urea residues are linked on separately at two different
stages in the synthetic process.

Properties. — Pure uric acid forms a white powder, which is made up
of small rhombic crystals, of more or less prismatic or tabular type. ^ Its
crystalline forms become very diverse in the presence of impurities,
and when it separates from the urine, the crystals, which are then always
coloured, take shapes which depend to a large extent upon the nature of
the pigment associated with them^ (FigS- 50 and 51).

In cold water it is very in-
soluble, only dissolving to the ex-
tent of about 1 part in 15,000.
A litre of boiling water takes
up about half a gramme. Ether
and alcohol do not dissolve it. It
dissolves in oil of vitriol without
decomposition, and from the solu-
tion a crystalline sulphate separates
on freezing the mixture. By this
process pure uric acid may be
obtained from contaminated speci-
mens, the sulphate being resolved
into its constituents when treated
with water.

It acts as a somewhat weak
dibasic acid, but forms three orders
of salts. _

1. The neutral urates, M'oU, have an intense caustic taste and are
very unstable. They are decomposed by carbonates and even by the
carbonic acid of the air. As they are only produced in the presence of
caustic alkalies, and cannot exist in the presence of carbonates, it is un-

1 A. E. Garrod.

Fig. 50. — Uric acid.


likely that they can, under any circumstances, occur as physiological
products.^ _

2. The acid urates or hiurates, M'HU, are the most stable of the com-
pounds of uric acid. They are prepared by dissolving the acid at boiling
heat in weak solutions of the alkaline carbonates, from which they
separate, after cooling, in stellar crystals. These and the foregoing salts
were first studied by Bensch and Allan." The acid urates are less
soluble than the neutral salts. _

3. The quadriurates, HoUjM'HU. — These hyperacid salts, for the ex-
istence and importance of which we have now satisfactory evidence, were
first described by Scherer,^ and (independently) by Bence Jones,* but
they have since been more carefully studied by Sir Wm. Eoberts.^
They are best prepared by boiling uric acid with dilute solutions of
acetate of potassium, and from solutions so obtained a quadriurate
separates, as an amorphous precipitate, or in crystalline spheres.^ They
are very unstable, and when treated with water they split up into
biurates and free uric acid. Owing to this instability it is impossible to
determine directly their solubility in water ; but they are probably less
soluble than the preceding order of salts, as a strong solution of a
biurate, when treated with acid-sodium phosphate, gives an abundant
precipitate of a quadriurate. From analogy we might expect the three
orders of salts, as described, to be in a descending series as regards

Condition of uric acid in the urine : its spontaneous se^jaration. —
Coloured indicators which are sensitive to free uric acid give no
indication of its presence, as such, in freshly -passed urine. Again, the
quantity of uric acid present is generally greatly in excess of what
would dissolve in a volume of water equal to that of the urine. The
presence of neutral salts, and also, according to Elidel,'^ of urea, enhances
this solubility, but not to a degree necessary for the retention of all the
urinary uric acid in solution. We are led to expect, therefore, that it is
present not as free acid but as a more soluble compound. Neverthe-
less, most urines will, on cooling and prolonged standing, deposit a
certain (and sometimes a large) proportion of their uric acid in a free

We have to explain, therefore, the nature of the original solution and
the cause of the subsequent separation. The view generally held till
recently, and still current with some authorities, is that the acid exists as
biurates ; and that these are slowly decomposed, with liberation of the
free acid, by the action of the acid phosphates, according to the following
simple reaction : —

MHU -H MH.,PO, = H^U + M2HPO,

From acid urines, however, the uric acid is frequently deposited, in
the first place, not as free acid, but in the form of urates, forming a
precipitate which has long been known as the " lateritious deposit."

A careful study of the chemistry of this deposit has led Sir William
Eoberts to conclude that the above equation does not rightly, or at least

1 Eoberts. 2 Ann. d. Chem., Leipzig, 1848, Bd. Ixv. S. 181.

" Neubauer ti. Vogel, "Analyse des Harris," 9th edition, S. 192.

^ Journ. Chem. Soc, London, 1862, voL xv. p. 8.

^ " Croonian Lectures," 1892. ^ Ibid.

■^ Arch./. cxjKr. lath. u. Pharmakol., Leipzig, 1892, Bd. xxx. S. 469.



does uot completely express the chemical mechanism of iiric-acicl solution
and precipitation.!

The urate deposit is amorphous, but on treatment with water it is
found to decompose, part of its uric acid being set free in crystalline
form and part going into solution (Fig. 51). But this is a propeity which
was stated above to be specially characteristic of the quadriurates, and
closer examination shows that the greater part of an amorphous urate
deposit does, in point of fact, consist of those hyperacid salts, and not
of ordinary biurates.

Roberts' view is that the quadriurate is the only physiological type
of uric acid salt, whether in blood or in urine.

Fig. 51. — Uric acid. — In the lower half of the figure the crystals are
shown as they separate when a quadriurate deposit is decomposed
with water.

In the normal acid urine, immediately after its secretion, all the uric
acid is in this form. But in aqueous solution the quadriurates are neces-
sarily in a state of unstable equilibrium, and tend at once to decompose
according to the equation —

(1) MHU,H2U = MHU + H2lJ;

half the uric acid being precipitated and the other half remaining in
solution as biurates. But the latter are in the presence of acid phos-
phates, and this fact again involves a condition of unstable equilibrium;
the following change occurring —

(2) MHU + MH2PO, = MHU,H2tJ-|-M2HPO,,

1 Roberts, loc. cit.


In fact, quadriurates are thus re-formed, and become subject to the
same influences as before. " These alternating reactions — breaking up
of quadriurates by water into biurates and free uric acid, and recomposi-
tion of quadriurates by double decomposition of biurates with mono-
metallic phosphate — go on progressively, until all the uric acid may be
set free."

The quadriurates, therefore, are of great importance in the chemistry of
urinary uric acid, and beyond all doubt form an intermediate step in the
liberation of the free acid itself. The evidence that they are the form in

Online LibraryE. A. (Edward Albert) Sharpey-SchäferText-book of physiology; (Volume v.1) → online text (page 83 of 147)