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

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water. The presence of urates must be borne in mind when the ammonium-
sulphate precipitate is being dealt with, as these yield a coloration with certain
proteid tests ; and again, may lead to error if ordinary mucin is to be identified
by its yield of a reducing body on boiling with acids, for uric acid itself
reduces copper solutions.

Aeomatic Substances.

In addition to hippuric acid, which, owing to its importance as a
nitrogenous constituent, we have treated specially, the urine contains
other substances belonging to the aromatic group ; that is to say,
substances the molecular structure of which contains the benzene
nucleus. Under normal circumstances each one of these is present in
very small amount, but collectively they are of importance. For our
knowledge of their chemistry in the urine we are largely indebted to
the initiative work of Baumann.

Like hippuric acid {q.v.), they are derived in part from the aromatic
constituents of the food, and they are all increased by a vegetable diet ;
but also, like hippuric acid, they partly arise from the breakdown of
proteids. In their derivation from the latter it is possible that tyrosine
in the bowel is an intermediate stage, as many of them are greatly
increased when that substance is given by the mouth.^ We cannot deal
with these substances in great detail, but the characteristic types
of compounds in which the aromatic nucleus is found in the urine
should be noted. They comjmse, mainly, simple hydroxyl-suhstitution
products of benzene, and carhoxyacids related to these. We shall also
consider in this section urinary indol and skatol, which are nitrogenous
aromatic compounds. Most of the substances to be dealt with are

^ Brieger, ZtscJir. f. pliysiol. Chem., Strassburg, 1878, Bd. ii. S. 256 ; Wolkow und
Baumanii, ibid, 1891," Bd. xv. S. 228.


excreted as conjugated sid-pludcs (see p. 631), but the carboxyacids are
only in part so excreted. Inosit, which belongs to this group chemically,
has somewhat different physiological relationships to the other aro-
matics of urine.

Phenol (C0H5.OH) and kresol (CH,.CeH^. OH).— Traces of carbolic
acid are present in urine physiologically, but the " phenol " of the normal
fluid consists, as a matter of fact, mainly of the homologous Izresol ; and,
of the isomeric forms of the latter, paixtkresol is the commonest. The
properties of this substance, however, closely resemble those of phenol
itself. The amount of phenol and kresol taken together may be upon a
mixed diet no more than some 30 mgrms. per diem. If the urine
be acidified and distilled, the distillate made alkaline, concentrated, and,
after concentration, neutralised, on the addition of bromine a whitish pre-
cipitate will appear, due mainly to the formation of the tribromphenols.

Pyrocatechin (orthodihydroxybenzene) and hydrochinon (para-
dihydroxybenzene) CoB.iiO'H.)^.- — Of these two isomeric substances
the former is a constant constituent of human urine in small
quantity ; the latter is found probably only under exceptional cir-
cumstances. The former is easily removed from the acidified urine
by shaking with ether; but its subsequent purification involves a
lengthy j)rocedure.^ It is a white crystalline volatile solid, easily
soluble in water. It gives a dark green coloration with ferric chloride,
which, on the addition of ammonia, becomes violet and afterwards
cherry -red.

Inosit. — This substance, from its sweet taste, was originally classed
with the sugars, and was known as " muscle sugar." It strictly belongs,
however, to the group of substances we are considering, as it is by com-
position hexahydroxybenzene (CH.OH)g. It appears in normal urine
with considerable frequency when polyuria is induced by diuretics or by
copious drinking. On the other hand, its appearance is not entirely
dependent upon the flushing of the tissues which such polyuria might
denote, as extreme polyuria is at other times not associated with
inosituria. It may occur in diabetes. Galloise found it in five out of
thirty cases.

It may Idc separated from the urine by precipitation with acetate of
lead. The precipitate is decomposed with hydrogen sulphide, the fluid
concentrated, and finally precipitated by admixture with a large bulk of
alcohol. The alcohol precipitate is dissolved in water, the solution
mixed with an equal bulk of spirit and poured into ether, which pre-
cipitates the inosit almost pure.

The substance forms crystals not unlike those of cholesterin. It is
optically inactive and does not ferment. It is said to yield sarcolactic
acid by the action of bacteria.

Of the aromatic carboxyacids the following have been identified
in human urine: — Parahydroxyphenyl-acetic acid Ol^.QJ^^ — CHg.COOH;
]Ktrahydroxyphenyl-23ropionic acid OH.CeH^ — CjH^.COOH ; dihydroxy-
'phenyl-acetic acid (0H)2 — CH2.COOH (the homogentisic acid of
Wolkow and Baumann) ; ^ and trihydroxyphenyl-jjropionic acid
(OH)3C6H2.C,H,.COOH (the uroleucic acid of Kirk).

In the urine of herbivora other analogous compounds have been

1 Cf. Halliburton, "Chemical Physiology and Pathology," 1891, p. 74.5.

2 Loc. cit., 1891, Bd. xv. S. 241. This is that isomeric acid which is related to hydro-


detected, and kynurcnic acid, a related substance, is an important consti-
tuent of dog's urine.^

Pathologically, the substances just described may become of considerable
importance. In carbolic acid poisoning many of them are excreted in greatly
increased amount; pyrocatechin and hydrochinon may be present in large
quantity, and then give rise, by their oxidation, to the peculiar coloration seen
in carholuria. In certain diseases other members of the group are increased,
and give rise to the phenomena of alcaptonuria. In this state the urine
develops, on standing, a dark colour, like that seen in carboluria ; and
Boedecker in 1861 isolated a substance which he termed alkapton, to the
oxidation of which he held the colour due. Alkapton Avas shown by Marshall
and Kirk to be impure uroleucic acid (vide supra). This latter substance,
however, is not wholly responsible for the coloration phenomenon. Wolkow
and Baumann - have recently shown that in a case investigated by them the
" alkaptonuria " Avas almost wholly due to the presence of homogentisic acid
{supra). Pyrocatechin is doubtless sometimes the cause. ^ Most hydroxy-
derivatives of benzene in alkaline solution develop a dark coloration on
exposure to the air* (cf. p. 630).

The quantity of phenol and kresol in the urine is increased in extreme
constipation, in obstruction of the lower bowel, in peritonitis, and in pyaemia
(cf. indoxyl, infra, and p. 631).

Indoxyl and skatoxyl. — These, although nitrogenous compounds,
are closely related to the substances just treated, and may fitly be
considered here.

Indoxyl (CuH^.NH.CH.C.OH). — The so-called urinary indican is
indoxylsulphuric acid. In normal urine on a mixed diet, the quantity
present is only from 5 to 20 mgrms. In herbivora the quantity
is much larger. It is absent from the urine of new-born children
(Senator). Indoxyl is derived from oxidation in the body of the indol
absorbed from the bowel, and its amount is increased, like that of the
urinary phenols, by all causes which lead to increased l^acterial decom-
position of proteids, in the intestine or elsewhere ; and by circumstances
which favour the absorption of the indol when formed (intestinal
obstruction, etc.). Skatoxyl (CgHgNOH) is derived from skatol (methyl-
indol), and accompanies indoxyl into the urine by parallel paths and
from kindred causes. Like indoxyl, it is present as a conjugated

By oxidation indoxyl forms indigo-blue and indigo-red, while skatoxyl
similarly yields red pigments. The consequent colour phenomena
which arise in the urine are discussed under the head of the pigments.

Carbohydrates and PtELATED Substances.

Normal urine contains small quantities of certain carbohydrates.
Under ordinary circumstances the physiological limit extends only to
a minute quantity of any one of these substances ; but, in the urine of
women during lactation, milk-sugar may occur in very considerable
amount, without departure from what must be considered physiological

^ For phenaceturic acid, see under hippiuic acid. - Loc. cit.

^ Cf. V. Jaksch, " KHnische Diagnostik," 4th edition, 1896, p. 415.

* The behaviour of the alkaline solution of pyrogallic acid used in photography will
be an example familiar to many.


The general chemistry of the carbohydrates is elsewhere discussed.
We shall deal only mth their relation to the urine : —

(a) Dextrose. — The question as to whether or not small amounts of
grape-sugar are excreted in the urine during normal health has been
much debated. It is needless to confuse the issue by an attempt to
define what is meant by " normal " urine. We may ask rather, Does
the urine of the average individual, living an ordinary Hfe, upon ordi-
nary diet, generally contain sugar ? There can be little doubt, in the
light of our present knowledge, that this question must be answered in
the affirmative.

Briicke ^ was the first (in 1858) to state that sugar is normally present in
human urine, and Bence Jones ^ was an early supporter of the view. For
some years, however, the question was treated as an open one, and in 1871,
Seegen, after careful study of the matter, decided that means were not then
to hand by which its presence could be proved with certainty. Pavy,^ in
1878, affirmed that it was certainly a normal constituent, and has always
maintained this position. Since then other observers (in England especially
Sh G. Johnson and G. Stillingfleet Johnson*) have stoutly mauitained the
contrary. The chief criticism of the earlier methods of demonstration which
gave positive results was that, while they dejDended upon reduction tests, they
did not eliminate the influence of other reducing substances. The creatinin,
uric acid, hippuric acid, and other aromatic constituents of the urine, all tend
to reduce salts of the heavy metals in alkaline solution. It is admitted by all
observers that normal urine exercises a reducing power on copper solutions,
which, if due to glucose, would indicate the j)i'esence of about 0*1 to 0"3 per
cent, of this substance. But it is equally admitted by all that a large part of
this reduction is due to the other substances mentioned above. The question
which has been at issue is as to whether any part of the reducing power is due
to sugar.

It is evident that we cannot rely alone upon reduction tests applied to
the original urine. The more accurate knowledge that we now posesss with
regard to the question has been obtained by three lines of investigation : —
(1) By the application of direct tests Avhich are unaffected by ' substances
other than sugar ; (2) by the use of methods which involve a preliminary
removal of interfering substances; and (3) by the employment of means
Avhereby the sugar itself is separated from the urine unmixed with the con-
stituents which lead to error.

1. Tlie phenylhydrazine test of Fischer and v. Jakscli has given positive
results in the hands of several observers when applied directly to normal urine. ^
The yellow crystals of phenylglucosazone may certainly be obtained from
urine containing as little as 0"1 per cent, of sugar (v. Jaksch). In my own
experience great care is generally necessary to secure unequivocal results in the
case of normal inline. As a crucial test, it suffers the disadvantage of yielding
crystals with the glycuronic acid compounds ; the amount of crystals obtain-
able from normal urine direct being in general too small for discriminating
tests to be applied to them. After the sugar has been previously isolated, the
reaction with phenylhydrazine is, however, of the utmost value as a confhma-
tory test {vide infra).

A colour reaction may be observed in normal urine, which is held by some
to be conclusive of the presence of sugar. This is the furfural reaction. A

^ Sitzungsh. d. Tc. Akad. d. Wissensch., Wien, 1858, Bd. xxix. S. 346.
- Journ. Chem. Soc. , London, vol. xiv. p. 22.
^ Ghiy's Hasp. Rep., London, voL xxi. p. 413.

■* See articles and correspondence in the Lancet, London, dnring July and August 1894.
5 Cf. E. Roos, Ztschr.f. physiol. Chem., Strassburg, 1891, Bd. xv. S. 523; A. H. Allen,
" Chemistry of the Urine," 1895, p. 89.


small quantity of /3-naplitliol dissolved in chloroform is added, and tlien some
strong sulphuric acid. The latter, by acting upon the traces of sugar ^jresent,
produces furfurol, which, with the /3-naphthol, gives a violet or carmine-red
coloration.^ This test is also affected by the presence of glycuronic acid.

2. By treating the urine Avith mercuric acetate, creatinin and the various
non-saccharine reducing substances are precipitated. G. S. Johnson has main-
tained that in the filtrate obtained after treating a normal urine in this way,
no sugar reaction can be observed. A. H. Allen has, however, obtained posi-
tive results. 2

3. By far the most satisfactory evidence is obtained by methods
capable of isolating any sugar that may be present. Moritz, by treating
5 to 6 litres of the urine of healthy men with lead salts and ammonia,
and by decomposing the precipitate so obtained w^ith sulphuretted
hydrogen (Britcke's method), was able to isolate a suljstance which gave
all the reactions of grape-sugar. It was fermentable with yeast, yielded
phenylglucosazone crystals, was dextrorotatory, and reduced alkaline
copper and bismuth solutions.^ Pavy, by a similar method, long ago
obtained a fermentable reducing body from normal urine, and he has
since extended his earlier results by showing that the substance yields

When solutions of carbohydrates are treated with benzoylchloride,
they yield a precipitate of insoluble compounds (esters) with benzoic
acid. Glycuronic acid gives no precipitate. Baumann has applied this
fact to the separation of urinary carbohydrates ; and, in the hands of
Wedenski ^ and Baisch,*^ the method has yielded very convincing results.
The last observer decomposed the benzoic esters he obtained from
normal urine, with alcoholic soda, and isolated, inter alia, a sugar which
gave, with phenylhydrazine, an osazone melting at the right temperature
for that of glucose. The product gave also all the other reactions of
dextrose. The quantity found varied from 0"08 to 0'18 grms. in the
twenty-four hours.

The evidence we have detailed leaves little room for doubt that
grape-sugar is a constituent of normal urine, and we may take the
figures just quoted from Baisch as the most accurate estimate we possess
of its amount. Pavy and v. Udransky found larger quantities, and
Seegen considerably less, but their methods are perhaps more open to
question from the quantitative point of view.''

Alimentary glycosuria. — It is certain that many healthy individuals, after
a meal rich in sugar, and especially after the consumption of an excessive
amount of sugar in solution — as in sweet wines and the like — excrete tem-
porarily quantities of sugar greatly in excess of the small normal constant we
have just discussed. The explanation of this is probably to be found in the
observation of Ginsberg,^ that when large quantities of sugar are present

^ Moliscli, CentralU. f. d. mecl. TVissensch. , Berlin, 1888, Nos. 34 and 49. Also Luther,
Chem. Centr.-Bl., Leipzig, 1891, Bd. ii. S. 90 ; v. Udransky, Ztsclir.f.fliy'iiol. Chcm., Strass-
burg, 1888, Bd. xii. S. 380.

''- Loc. cit., p. 19.

^ Deutsches Arch. f. klin. Med., Leipzig, 1890, Bd. xlvi. S. •252. A complete review of
the earlier literature will be found in this paper.

■* "Physiology of the Carbohydrates," 1894, p. 180 et seq.

^ Ztschr. f. pMjsiol. Chem., Strassbnrg, 1889, Bd. xiii. S. 122.

« Ibid., 1894, Bd. xviii. S. 193 ; 1895, xix. S. 348 ; xx. S. 249.

' For a criticism of Briicke's lead-precipitation method, see Colls, Joiirn. Physiol., Cam-
bridge and London, 1896, vol. xx. p. 109.

8 Arch.f. d. ges. Physiol., Bonn, 1889, Bd. xliv. S. 306.

VOL. I. 39


in the bowel, some may be absorbed, not by the ordinary path of the capil-
laries and portal vessels, but by way of the lacteals and thoracic dnct, thus
escajDuig the influence of the liver. The percentage of sugar m the blood is
thereby increased, and the excess is excreted by the kidneys. A distinction
between such cases and those in which diabetes exists, is seen in the fact that
the " alimentary glycosuria " is not produced by starchy foods, however large
the quantity taken, but only by excess of ready-formed sugar in the diet.^
According to Moritz, dextrose, laevulose, cane-sugar, and probably milk-sugar,
may all appear unaltered in the urine when severally taken by the mouth in
considerable quantity {e.g. 200 grms.). Such alimentary effects last from
three to six hours.-

Patliological glycosuria. — The great increase of dextrose in the urine
of diabetes is a familiar phenomenon. Its excretion may range in this disease
from quite small quantities up to 500 or 600 grms. per diem. The morning
urine is apt to contain least sugar ; that passed three or four hours after a
meal generall}'' contains most.

In other diseased conditions, quite apart from diabetes, a special tendency
has been observed to the occurrence of an alimentary glycosuria ; gout,
exophthalmic goitre, and certain nervous diseases may be instanced. An
increase of reducing substances, almost certainly consisting at least in part of
dextrose, is said to be found in the urine of some pyaemic conditions. As the
effect of certain drugs and toxic substances, such as chloral, chloral amide,
morphine, hydrocyanic acid, turpentine, and carbon monoxide, the urine
commonly reduces copper solutions ; but in most of these cases the reduction
is due to conjugated compounds of glycuronic acid, and not to dextrose
{vide p. 613).

The detection and estimation of dextrose, which, as we have seen, have
proved difficult problems in the- case of the minute amount normally present
in urine, become easy when the increased amount excreted in disease is to be
dealt with. The methods used depend upon the reducing power which the
sugar exerts upon metallic salts, or upon certain coloured organic substances,
and these may be checked by the fermentation of the suspected urine by
means of yeast, by the indications of the phenylhydrazine test, and again by
the use of the polariscope. Of reduction tests a great immber have been
proposed ; we shall here refer to two only. The well-known Fehling's test
consists of a solution of copper sulphate of definite strength, mixed with caustic
alkali and alkaline tartrates (Rochelle salt). The presence of the last pre-
vents the precipitation of cupric oxide when the solution is boiled by itself,
but allows the precipitation of yellow or cuprous oxide when reduction has
occurred from the action of the sugar. The reduction may be observed,
after boiling the liquid, if the urine contain not less than 0*2 per cent, of
dextrose. If less than about 0'5 per cent, be present, no precipitation occurs
until after cooling, when the liqiiid becomes opaque, and of a greenish colour.
With larger amounts a definite precipitate of a yellow or red colour is seen,
immediately after heating the test with a small proportion of the urine.
Nylander^s solution has some advantages over Fehling's, in that it is much less
affected by creatinin, urates, and reducing bodies other than sugar (vide supra).
It is a modification of the bismuth test, origmally suggested by ElJttcher, and
is prepared Avith the same reagents as Fehling's solution, biit with the sub-
stitution of basic nitrate of bismuth for the copper sulphate. On boiling this
solution with urine contaming sugar, the liquid turns black. The reaction is
easily seen if 0"1 per cent, or upwards of dextrose is present.

Both solutions are reduced by lactose and by glycuronic acid; but the
former of these substances can ouly be present under special circumstances

1 Cf. Xeumeister, " Lebrbuch der pbysioL Cheni.," Tb. 2, S. 306.
-Moritz, Centralhl. f. Iclin. Med,. Bonn, Bd. xii. Ko. 28.


{infra), while the latter is never present in the urine in amounts large
enough to lead to confusion with glycosuria in the pathological sense;
except when quite special substances have been taken by the mouth. But
in order to make the identification of glucose more certain, we may confirm
the results of a reduction test by means of yeast fermentation. Lactose and
glycuronic acid do not ferment. The urine should be placed in a test-tube
so as completely to fill it, and the tube inverted over a basin containing a
further quantity of the urine. After ascertaining that no air is present, a
small piece of pressed yeast is passed under the inverted tube, and the latter
secured in position with a clamp. The tube is then allowed to stand at a
temperature of 25° to 30° C. In twelve hours, if dextrose be present in
quantity, a notable amount of carbon dioxide will have collected in the upper
])art of the tube. The fermentation test is very conclusive, but it is not easily
obtained when less than 0*5 per cent, dextrose is present. With phenyl-
hydrazine, however, as already stated, urines containing as little as O'l per
cent, will yield easily recognisable crystals of phenylglucosazone. The fact
that Isevulose also ferments with yeast, and yields an identical glucosazone, is
of little importance in practice ; this sugar is rarely present {infra), and except
under special circumstances it is unnecessary to distinguish it from dextrose.

For the estimation of dextrose, modifications of the various tests just
described are employed. We may ascertain, for instance, how much of a
given specimen of urine is required to precipitate all the copper from a
measured quantity of standardised Fehling solution. Or, with greater con-
venience, we may employ the modified copper test known as Pavy's solution.
This contains a large excess of ammonia, in addition to the ordinary constitu-
ents of Fehling's test. In ammoniacal solution cupric salts are blue, but
cuprous salts are colourless. By noting, therefore, the amount of the urine
(diluted, if necessary, to a known bulk) which is necessary to decolorise a
given quantity of the standard Pavy's test, we obtain a measure of its reducing
power, and so of the dextrose present. Again, we can adapt the fermentation
test to quantitative purposes, by measuring the COo produced from a definite
quantity of the urine, or by ascertaining the diminution in the specific gravity
of the fluid which follows the destruction of the sugar by the yeast. Lastly,
we may employ the polarimeter, which indicates the percentage of dextrose by
the number of degrees through which a polarised ray is turned to the right
on passing through a layer of urine of determinate depth. A drawback to
the use of this instrument, when applied to the urine, arises from the fact
that other substances may be present which are optically active. ^

(b) Leevulose. — The occurrence of Isevulose in normal urine has not
been observed ; but in certain cases of glycosuria it is said to be present.
Kulz^ separated from the urine of a diabetic a Isevorotatory sugar,
which possessed all the properties of ordinary Isevulose, except that,
unlike the latter, it v^as precipitated by basic acetate of lead. When
lievulose is given by the mouth in diabetes, it can be utilised in
metabolism more readily than dextrose, and within certain limits of
administration it is not excreted in the urine. Beyond these limits,
however, it is eliminated partly unaltered and partly as dextrose.^

(c) Lactose. — That a reducing substance is apt to appear in the urine
of women during the period of lactation, was first observed by Heller as
far back as 1849 ; and F. Hofmeister,* in 1877, showed definitely that

^ Details of all these processes will be found in practical handbooks.

"- Ztsclir. f. Biol., Mllnchen, 1890, Bd. ix. S. 228. References to the earlier literature
will be found in this paper.

» Of. Haycraft, ZtscM. f. ]]hysiol. Chevi., Strassburg, Bd. xix. S. 137 ; Hale AVhite,
Guy's Hasp. Bep., London, 1893, p. 133.

^* Ztschr.f. pliysiol. Chem., Strassburg, 1877, Bd. i. S. 104.


this was railk-sugar. Only lately, however, has it been recognised that
lactosuria is an almost constant phenomenon during lactation. Even

Online LibraryE. A. (Edward Albert) Sharpey-SchäferText-book of physiology; (Volume v.1) → online text (page 86 of 147)