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when the conditions are altogether favourable and normal, it is seldom
that the sugar is not present in the urine at some portion of the period.
When any interruption to the natural removal of the milk occurs, the
amount may be very considerable.

The phenomenon is easily understood when it is remembered that
the lactose excreted into the blood from the mammary gland does not
come under the normal influence of the liver. Eecent researches,
indeed, indicate that milk-sugar cannot in any case act as a precursor
of glycogen, until it has been inverted. When lactose is taken by the
mouth, this inversion occurs before or during absorption from the bowel.

The complete identification of lactose in the urine is difficult, unless it be
first isolated by processes too lengthy to be described here. But if the urine
exhibit the following characters, the presence of lactose is established almost
without the possibility of doubt. It should reduce copper and bismuth solu-
tion ; but, with the fermentation test, it should give negative results for the
first twenty-four hours of the experiment, and it should give no definite crystal-
line precipitate with the phenylhydrazine test when this is directly applied. ^
On the other hand, after boiling with 5 per cent, sulphuric acid for a short time,
the urine should, if first neutralised with ammonia, give the phenylhydrazine
test readily; crystals of dextrosazone should be thus obtained, and, with
proper precautions, galactosazone crystals may be also distinguished. Although
the lactose is converted by the mineral acid into dextrose and galactose, a
fermentation is not always to be obtained after treatment, as the large amount
of sulphate, which is present after neutralising the acid, interferes with the
growth of the yeast. If the reducing power of the urine be estimated, this
should be found increased after boiling with mineral acid, but unaffected by
boiling with citric acid.

(d) Pentoses — -Xylose, arabinose (C5H10O5). — Ebstein,- Salkowski,^ and
others have observed the presence of 5 -carbon sugars in the urine.
They are generally, when present, derived from the food, and then pro-
bably arise from certain fruits, especially cherries and plums, which
contain either pentoses or a precursor of these sugars, the so-called
" fruit gum." The pentoses are apparently assimilable with difficulty.
Under exceptional circumstances, it seems possible that they may
arise in the organism, as the result of disordered metabolism. It has
been found that a certain proteid, derived from the pancreas, yields
pentoses when boiled with acids, and some such substance may be the
source of pentosuria.

The pentoses give a red coloration when treated with strong hydrochloric
acid in the presence of phloroglucin (Tollens' reaction). Glycuronic acid,
however, behaves similarly. They reduce copper solutions, and yield an
osazone after somewdiat prolonged warming with phenylhydrazine, but they
do not ferment.

(e) Isomaltose. — When the mixture of carbohydrates obtained from
normal urine by precipitation with benzoylchloride is fermented with
yeast, so that all the dextrose present is destroyed, there remain small

^ Lactosazone does not crystallise readily, except from pure solutions of the sugai'.
- Virchow's Archiv, 1892, Bd. cxxix. S!! 401 ; cxxxii. S. 368.

2 Cenlralhl. f. d. med. Wisscnscli., Berlin, 1893, S. 193 ; Berl. klin. JFchnschr.,
1895, No. 17.



ANIMAL GUM— GLYCURONIC ACID. 613

quantities of a sugar, which, though not fermentable, gives a well-
crystalhsed osazone, and reduces Fehling's sohition. According to the
researches of Baisch,i the properties of this substance agree with those
of " isomaltose."

(f) Animal gum. — The third and remaining carbohydrate which
separates from normal urine when this is treated with Ijenzoylchloride,
is a dextrin-like substance, in all probability identical with "animal
gum." This was first isolated from urine by Landwehr,^ who took
advantage for this purpose of the insolubility of its copper compound.
Its presence has been confirmed both by Wedenski and Baisch, who
employed the benzoylchloride method.

The substance does not reduce metallic salts, but, on the other hand,
on boiling with mineral acids, it yields a derivative which will reduce
Eehling's and Nylander's reagents freely. Simultaneously it yields
(like many other carbohydrates and certain of the aromatic constituents
of the urine) with acids a brown flocculent precipitate of the " humous
substances " of v. Udransky.'^ It is due to the presence of this substance
that the reducing power of most urines is increased after boiling with
mineral acids.

(g) Glycuronic acid. — The chemical relationship of this acid to the
glucoses is seen by a comparison of their respective formukc : —

CH^HOCCROHXCHO COOH.(CH.OHXCHO

(glucose) ■ (glycuronic acid)

It is derived from these sugars by oxidation of the primary alcohol
group, CH2.OH, to the carboxyl group, COOH. It is at once, therefore,
an aldehyde and an acid. As an aldehyde, it reduces copper solutions.

It should be understood that glycuronic acid is never a constituent
of normal urine in appreciable amount, nor does it appear as the result
of pathological processes in the ordinary sense. Its presence almost
always depends upon the ingestion of special substances, which are for
the most part foreign to ordinary foodstuffs ; and, when excreted, it is
" conjugated " with these, or with derivatives of these.

It is apparently an intermediate product in the metabolism of
carbohydrates, which, normally, becomes fully oxidised in the body ;
but which, when conjugated with the exceptional substances referred to,
escapes oxidation, and appears in the urine, just as the easily oxidisable
glycin is protected by conjugation with benzoic acid and appears as
hippuric acid.

Some of the substances which form these conjugated compounds
with glycuronic acid, are those which ordinarily form conjugated or
ethereal sulphates (cf. pp. 606 and 631), especially the aromatic hydroxy-
compounds. Phenol- indoxyl- and skatoxyl-glycuronic acids and many
analogues have been described in the urine. But apparently the sulphate
conjugation is the more constant process, and it is only when the above
substances are present in very large amount that their glycuronic con-
jugates appear in addition to their sulphuric acid compounds, — in
general, only when they, or their precursors, are given abundantly by
the mouth for the purposes of experiment.

Of more practical importance are those conjugated compounds of

^ Baiscli, Ztschr. f. physiol. Cliem., Strassburg, Bd. xx. S. 249.

2 Centralhl.f. d. med. Wisscnsch., Berlin, 1885, S. 369.

^ Ztschr. f. physiol. Chem., Strassburg, 1888, Bd. xii. S. 33.



6 14 THE CHEMISTRY OF THE URINE.

glycvironic acid with members of the fatty group of alcoliols, which are
excreted after the use of certain common drugs. Thus, when chloral
hydrate is being taken, trichlorethylglycuronic acid (urochloralic acid) ^
is often found in the urine, and analogous compounds arise after the
administration of butylchloral hydrate and chloroform.

All these compounds are lasvorotatory, though glycuronic acid itself
is dextrorotatory ; many of them, urochloralic acid for instance, reduce
bismuth and copper solutions freely, and their presence may therefore
lead to error in testing for sugar ; but they are not fermentable. They
spHt up with varying degrees of ease into glycuronic acid and the
conjugated substance, either by boiling with mineral acids, or when
heated with water in sealed tubes ; some {e.g. phenolglycuronic acid)
decompose when boiled with water alone.

Glycuronic acid itself is a syrupy substance, soluble in water and
alcohol; but when its aqueous solutions are boiled or evaporated, it
loses water, and forms a crystalline anhydride which is insoluble in
alcohol.

To separate the urinary glycuronic compounds, a large quantity of urine is
precipitated with acetate of lead, and the precipitate decomposed with
sulphuretted hydrogen. After filtering, barium hydrate is added to the
solution. The sulphates and phosphates thus precipitated are filtered ofi^ and
alcohol added to the filtrate, whereupon the barium salts of the conjugated
glycuronic acids crystallise out.^

Other Organic Compounds.

Oxalic acid. — Small quantities of oxalic acid (C00H)2 are present
in all specimens of urine, about 50 mgrms. being the average for the
day's excretion. Much of this may arise directly from preformed
oxalates ingested with the food, as all vegetable food contains traces of
these salts, and direct experiment has shown that they are susceptible
of but very incomplete oxidation in the body.^ But oxalic acid does
not disappear from the urine when pure flesh-food is taken, nor even
during starvation;* it would thus seem certain that it can arise
from proteid metabolism. It is, in certain cases, very largely increased
in amount, from causes which are not clearly imderstood. Some
authorities hold that these cases of " oxaluria " depend always upon
excess of preformed oxalates in the diet ; but no one who has observed
the marked tendency to increased oxalate excretion in diabetes, or the
way in which, in some cases of glycosuria, a temporary decrease in the
sugar may be associated with an increase of oxalates, can doubt that it
may arise also from incomplete oxidation of carbohydrates.

Oxalate of ealcium frequently separates from the urine to form a
crystalline deposit. It mostly takes the form of the so-called
" envelope crystals," but may appear as dumb-bells, and is often seen as
clear ovoids (Fig. 56). It is responsible for the formation of a variety of
urinary calculus.

1 This was the first of tliese substances to be described, vide Muscuhis and v. JMering,
Bcr. d. deutsch. chem. Gesellsch., Berlin, 1875, Bd. viii. S. 662.

2 Of. Ashdown, Brit. Med. Journ., London, 1890, voL i. p. 169.

^ Gaf^lio, Arch. f. CKjier. Path. u. PharmalcoL, Lei])zig, 1887, Bd. xxii. S. 246.
■^Marfori, Jahresh. ii. d. Fortschr. d. Thier-Chcm. , Wiesbaden, 1892, S. 72.



ACIDS AND HYDROXYACIDS.



615



To demondrate the presence of oxalic acid, and to estimate its aiuoiint, a
litre of urine should be treated with calcium chloride and ammonia, and
afterwards made acid with acetic acid. After twenty-four hours' standing,
the crystalline precipitate, which contains uric acid crystals mixed with
calcium oxalate, is filtered off and treated with dilute hydrochloric acid. The
oxalate dissolves, and is reprecipitated, after filtering off the uric acid, by the
addition of ammonia. At a dull red heat it is converted into calcium
carbonate, and may be weighed as such.




Fig. 56. — Calcium oxalate.

Acids and hydroxyacids of the fatty series, with derived
substances. — Normal urine contains minute quantities of the volatile
fatty acids, especially acetic, but also formic, propionic, and butyric
acids.^ They do not, as a rule, amount collectively to more than some
50 mgrms. in the day's excretion, and they arise doubtless from the
bacterial decomposition of carbohydrates and proteids in the lower
bowel. Fatty acids of low atomic weight, such as the above, are less
easily oxidised in the organism than are those of greater complexity.^

The amount is considerable in the urine of herbivora, and in man it is
increased by many diseases, especially by such as lead to increased decomposi-
tion in the bowel, or to prolonged constipation.

When a specimen of urine undergoes ammoniacal fermentation, the
volatile acids are increased at the expense of the carbohydrates it contains.^

These acids are obtained from the urine by distillation with phosphoric



iCf. V. Jakscli, Ztschr. f. physiol. Chenu, Strassburg, 1886, Bd. x. S. 536. Tlie
earlier literature is here summarised.

-C. Scliotten, Unci., 1883, Bd. vii. S. 375.
^Salkowsld, iUd., 1889, Bd. xiii. S. 264.



6i6 THE CHEMISTRY OE THE URINE.

acid. They are found in the distillate so obtained, together with traces of
hydrochloric and benzoic acids, phenol, and acetone.

Sarcolactic acid is not a normal component of human urine, but it appears
in many diseased or abnormal conditions of the body, of which it may be said
generally that they involve either a suspension of normal hepatic functions or
interference with the proper oxidative processes of the body.^ It was first
observed in the urine of phosphorus poisoning, and of acute yellow hepatic
atrophy,^ and may be always demonstrated in these conditions. It is found
also after slow asphyxia ; in poisoning by carbon monoxide, in prolonged
anaemia, and shortly before death in very many diseases. That it may appear
after prolonged and severe exercise, is doubtless explained by the fact that
oxidation in the body has not kept pace with the increased production of
lactates in the muscles. ^

The three closely related substances, /^-hydroxyhutyric acid, acetacetic
acid {diacetic), and acetone rise to importance only in diabetes, but small
quantities of the last may be found in normal urine, and all may be increased
in disease apart from glycosuria. The following equations show the relation
which obtains between them : ^ —

CH3CH(OH).CH2.COOH + = CHo.CO— CH^.COOH + H2O

(/3-hydroxyl)utyric acid) (diacetic acid)

CH3.CO— CH.^.COOH = CH3.CO.CH3 + CO2

(diacetic acid) (acetone)

The first only appears in the urine in conjunction with the others, but
either of the two latter may be found alone. Large amounts of all three may
be found in diabetes ; of the hydroxy-acid many grammes may be passed in
the day.

The presence of diacetic acid may be demonstrated by making the urine
acid with sulphuric acid, and shaking with ether ; the latter, which extracts
the substance, is then transferred to another vessel, and is shaken with a weak
aqueous solution of ferric chloride, which, if acetacetic acid was present in the
urine, becomes of deep burgundy wine colour.

In testing for the liydroxybutyric acid, advantage is taken of the fact that
it yields a volatile derivative, a crotonic acid, on distilling with sulphuric acid.
This substance crystallises out from the distillate of the urine, and may be
identified by its melting point (72° C). The urinary liydroxybutyric acid is
laevorotatory.

Acetone is identified in a urinary distillate by first adding a few drops of a
solution of sodium nitroprusside, and then caustic alkali, whereupon, in the
presence of acetone, a fine cherry-red colour is jDroduced. Acetic acid subse-
quently added in excess changes the colour to purple {LegaVs test).



The Colour of Uiiine and the Chemistey of the Pigments.

It is a familiar fact that, under physiological conditions, the urine
may be almost colourless, or may exhibit tints varying from a pale
straw yellow, through deep orange, to reddish brown. In its commonest
condition it is yellow. Pathologically, the colour may undergo variations
wider than those seen in health.

^ Cf. Araki, Ztschr. f. physiol. Chem., Strassburg, 1895, Bd. xix. S. 422, with reference
to previous jjapers by tliis author.

^ Schultzen u. Riess;, Chem. Cenir.-BL, Leipzig, 1869, S. 681.

2 Colasanti and Moscatelli, Arch. f. exiicr. Path. u. Pharmakol., Leipzig, 1890, Bd.
xxvii. S. 1.58.

* Cf. Minkowski, ihid., 1893, Bd. xxxi. S. 183.



THE COL O UR OF URINE. 6 1 7

An effort has been made to refer the varymg degree of pigmentation
to a standard colour scale, so that the condition of a given specimen, as
regards colour, might be quantitatively expressed. But much difficulty
intervenes, in that variations may be due, not alone to differing amounts
of a single colouring matter, but to independent and quite irregular
variations in at least three or four. The endeavour to attain to quanti-
tative precision has on this account proved unsuccessful in practice.^
We may content ourselves with speaking of physiological urine as pale,
normal, or high-coloured respectively, and assist the description by
comparison with other substances of familiar appearance (" straw-
coloured," " sherry-coloured," etc.).

Pale urine is usually of low density, and contains a small proportion
of solid matter. It results from all causes which promote a copious
flow of fluid from the kidneys, such as free ingestion of liquids, a check
to the cutaneous transpiration (as from the effect of cold), and emotional
excitement.

High-coloured urine is generally of high density, and is excreted
when the transpiration from the skin is more than usually free, or
under conditions of high metabolic activity. After a full meal the
urine is often at once copious and of full colour.

In general the amount of pigment rises with an increase in the con-
stituents excreted by the renal epithelium, and not with the glomerular
excretives. The depth of colour may be affected by the reaction of the
urine ; other things being equal, an acid urine will show a darker tint
than one that is alkaline.

Examined directly by means of the spectroscope, fresh normal urine
is found nearly always to show no definite absorption-band ; a diffuse
absorption of the more refrangible rays being alone evident.

But by the aid of the spectrophotometer ^ we may measure tlie amount of
light absorption in any region of the spectrum apart from the presence of
actual bands. When light passes through urine, the amount of absorption
increases progressively from the mid-red to the violet.

Suppose the absolute absorption at any Uno points in this region of
spectrum be measured ; say in the neighbourhood of the Fraunhofer lines, E
and F respectively. If in any one specimen of normal urine the absorption
near F is found to be twice as great as that near E, then if the urine contained
but one pigment, this same ratio would be found in any other specimen. The
absorption at F would in all cases be double that at E. For, clearly, the
dilution of an individual pigment would decrease the absolute absorption
throughout the spectrum, but would leave the relative absorption at any two
points unaffected; similarly, concentration would increase the absolute, but
would nowhere affect the relative, absorption. But different specimens of
normal urine do not agree in this way. One urine may show more relative
absorption (say) in the mid-green, another more in the blue. This can only be
due to the fact that more than one pigment is concerned. ^ Although it yields
no definite bands, the spectroscopic properties of fresh normal yellow urine
thus indicate some complexity in its pigmentation ; but the same experimental
evidence indicates nevertheless that no more than one pigment is usually
present in a relatively large amount.

^ By the use, however, of Lovibond's tintometer, the colour of urine under varying
circumstances may be very exactly imitated, and expressed iu terms of a scale.

^ See this textbook, article "Htemoglobin," p. 213.

^ This argument only holds for colouring matters which do not undergo dissociation in
solution.



6i8 THE CHEMISTRY OF THE URINE.

The pigments of the urine have long received attention and have
been the subject of many laborious researches ; but, owing to the great
difficulties they present to the investigator, our knowledge of the
chemistry of most of them has remained indefinite. These difficulties
arise from various causes. Pigment metabolism appears to be always
of a highly conservative nature. The colouring matters found in the
epidermal structures of animals, serving for ornament, protection, or
other purposes, are almost always present in strikingly small quantity ;
and those which are purely excretory leave the body in equally small
proportionate amount.

The highly developed optical activity of these substances, which has
led us to group them together in a special class as " pigments," at the
same time gives to them a prominence in various phenomena, dispro-
portionate to the actual quantity in which they are present. The
urinary pigments are (at least, under normal conditions) quite minute
in amount, and this fact is the primary difficulty in the path of chemical
investigation. As Bunge has written, many endeavours have resulted
merely in applying Greek and Latin names to substances which have
been obtained in quantity too small for proper investigation.

The extremely delicate indications of the spectroscope have been of
the greatest assistance in overcoming this fundamental difficulty, and
our knowledge of pigments has been much extended by its use. But
evidence so gained has to be checked and assisted by other methods. A
complex spectrum may indicate a mixture of substances ; but it may,
with equal probability, be due to one alone. A mixture, on the con-
trary, may show Ijut a single absorption-band, for the reason that of the
pigments present one alone extinguishes light in a specific region.

It is therefore easy, by a mere qualitative use of the spectroscope,
to mistake a mixture for a chemical individual. On the other hand,
very slight variations in the physical condition of a pigment, or a
minute change in its molecular constitution, may produce a great
effect upon its spectrum, and, unless we are aware of these conditions,
we may be led to see wide differences where chemically there is little or
none.

When, again, endeavours are made to isolate pigments by chemical
means, the great instability which they exhibit as a class is apt to lead
to error. So often has this danger been overlooked, that we are
compelled to attach' no importance, beyond what accrues from historic
interest, to much of the work which has been done on this problem.

It is of prime importance, when we endeavour to obtain these
unstable substances in their integrity, that the use of highly active
reagents should be avoided.

We shall deal only with the pigments of which we have comparat-
ively accurate knowledge ; but it may be safely asserted that the four
substances now to be described form the basis of urinary chromatology.
These are urochrome, urobilin, uroerythrin, and hccmatoiwrphyrin. Other
pigments exist, and some have doubtless yet to be recognised, but they
are exceptional, or take but very small share in the coloration of the
urine.

Preformed pigments of normal urine— (a) The essential yellow
pigment, urochrome. — In 1864,Thudichum gave the name of urochrome
to preparations obtained from normal urine by complicated processes of
extraction. Tliudichum's products undoubtedly contained a large pro-



PREFORMED PIGMENTS OF NORMAL URINE. 619

portion of the substance we are now to describe, but they were mixed
with urobilin and with decomposition products.

To A. E. Garrod ^ we owe a process for the extraction of the essential
yellow colouring matter, which is beyond reproach in its avoidance of
destructive reagents. It yields a product entitled to be considered, with
a large degree of probabihty, as a chemical individual.

Followuig Garrod, we shall describe this pigment under the name of
urochrome — a name eminently fitted for a substance which is the prime
cause of the famihar colour of urine, but in the use of which we nmst
avoid historical confusion.

Between the urochrome of Garrod and that described thirty years
earlier there is the difference between presumptive chemical individual-
ity and almost certain admixture. It should be stated, however, that
Thudichum still holds the pigment described by him to be a definite
substance, and has recently investigated certain of its reactions, which he
believes indicate for it the combined characters of an alcohol and a base.

Separation of urochrome (Garrod). — The urine is saturated with, crystals
of ammonium sulphate, and, after standing, is filtered {vide infra., " Separation of
Urobilin"). The filtrate, which is still almost as highly coloured as the original
urine, is shaken with alcohol. The latter solvent separates rapidly from the
saline mixture, and is seen to Avithdraw a large proportion of the colouring
matter. Kepeated extraction removes practically all. The alcoholic solution
is diluted with a large bullv of water, and the mixture again saturated with
ammonium sulphate ; by this procedure the alcohol is again made to separate
from the water, carrying the pigment with it, and a satisfactory washing of
the original extract is secured. This second alcoholic solution is now made
just alkaline with ammonia, and evaporated to dryness ; the residue is
extracted once or twice with acetic ether, which removes certain impurities,
and is again dissolved in strong alcohol. Somewhat prolonged digestion is
necessary at this stage, as the solubility in alcohol is decreased Avhen the
pigment has once been taken to dryness. Finally, the alcohol is concentrated



Online LibraryE. A. (Edward Albert) Sharpey-SchäferText-book of physiology; (Volume v.1) → online text (page 87 of 147)