A STUDY

BY

M.

(From
the Laboratories
cine of the University

E.

NYLANDERS

REHFUSS

AND

of Physiological
of Pennsylvania

REACTION.
P.

B.

HAWK.

Chemistry
of the Department
of Mediand the University
of Illinois.)

for publication,

January

21, 19 IO.)

In connection with our examination into the claims of Bechhold

as to the inhibitory
influence of mercury and of chloroform upon
Nylanders
reaction,2 we made a careful study of this reaction.
We investigated
the test from five different
standpoints
as
follows:
(a) Most satisfactory
method of performing
the test;
(b) its delicacy; (c) the influence of temperature
upon the reaction; (d) interfering substances : (e) its clinical value.
Method of Performing
the Test. There has been considerable
controversy
comparatively
recently as to the proper manner in
which Nylanders
test should be performed.
PAtigers claims, as
the result of a series of tests in which the urines under examination were boiled in a water-bath for 15-30 minutes, that the test is
useless inasmuch as more than one-half the normal urines give
a positive reaction.
Hammarsten4
defended the test against
Pfliigers attack.
He performed the test by boiling over a free
flame, for 2-5 minutes and claimed that when performed in this
way that the test was most satisfactory
and served to detect
small quantities of reducing material.
He calls attention to the
fact that Pfliiger boiled his tests for a period which is much too
long and asserts that the conditions are entirely different when
we boil 2-5 minutes over a free flame from those in force when
we boil 15 to 30 minutes on a water bath.
Hammarsten
says
it is not hard to understand
why normal urines might appear
1 Bechhold:
Zeitschr.
f. physiol.
Chem., xlvi, p. 370, 19 06.
1910.
2 Rehfuss
and Hawk:
This Joural,
vii, p. 267,
3 Pfltiger:
Arch. f. d. ges. Physiol.,
cxvi, pp. 265 and 533,
4 Hammarsten:
Ibid.,
cxvi, p. 517, 1907.
273

1907.

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(Received

OF

274

A Study

of Nylanders

Reaction

to give a positive

1 We used the following

formula
subnitrate;
4 grams Rochelle
salt;

for making
the reagent
: z grams bismuth
IOO cc. IO per cent potassium
hydroxide.

Downloaded from http://www.jbc.org/ by guest on January 19, 2016

reaction when subjected to Pfliigers manipulation.

Pfliiger, in criticising Hammarstens
procedure says that
heating over a free flame cannot fail to give gradations
in heat
and that only that side of the tube in immediate contact with
the flame can be perfectly heated. He believes that moreuniform heat is secured through his practice of using the waterbath.
In his tests upon the inhibitory action of mercury and of chloroform Bechhold instituted
a uniform heating for five minutes upon
a boiling water-bath.
After making a series of comparative
tests
of the methods proposed by Pfliiger, Hammarsten,
and Bechhold
we are forced to the conclusion that the five minute boiling period
on the water-bath
yields most satisfactory
results.
Therefore
we have adopted that procedure in all our work.
We have been
in the habit of using a large porcelain-lined
water-bath,
the bottom of which forms an admirable background
for observing and
comparing the various color changes.
The constancy
of condition, the fact that several tests may be made simultaneously,
together with the very material shortening of time seem to us sufficient reasons for using this modification.
In making the test 5 cc.
of the urine under examination are placed in a test-tube, 0.5 cc.
of Nylanders
reagent is added and the tube placed in a boiling
water-bath
for five minutes.
The tube is then removed and examined before a white background.
A black end-reaction
indicates the presence of reducing sugar in the urine under examination.
The Delicacy
of the Test.
Inasmuch as there is such great
diversity of opinion as to t.he delicacy of Nylanders test we considered it worth while to investigate this point.
Certain textbooks state that the test will detect sugar when present in a concentration of 0.025 per cent whereas other books set values of
0.05 per cent and 0.1 per cent asthe delicacylimitsforthereaction.
We first tested the delicacy of the reaction when applied to
aqueous solutions of dextrose as well as to normal urines to which
weighed amounts of dextrose had been added. Many tests of this
sort were made, the results from one series of aqueous solutions

M. E. Rehfuss

and P. B. Hawk

275

1 Daiber:

Corresp.

Schweizer

Aerzte, xxiv, p. 38.

Downloaded from http://www.jbc.org/ by guest on January 19, 2016

being recorded in Table I, p. 281.

Inasmuch as a positive reaction is indicated by an actual darkening of the solution following
the reduction of bismuth it will be seen that beyond 0.08 per cent
the test is no longer available for the detection of dextrose.
The
yellow end-reaction
obtained in solutions more dilute than 0.08
per cent is due to the action of the potassium hydroxide upon the
Solutions of lzevulose, maltose
sugar i. e., Moores reaction.
and lactose were examined in a similar manner.
The limit of
accuracy in the case of lzevulose was found to be 0.07 per cent,
whereas the values for maltose and lactose were the same as that
determined for dextrose.
The Eifect of Temperature upon the Reaction.
In these tests IO
cc. of a I per cent solution of dextrose were treated with I cc. of
Nylanders
reagent and the tube placed on a water-bath
at some
constant temperature
for a definite period, careful record being
made of the period of time necessary to secure the typical endreaction as well as certain definite intermediate
color changes.
The data from these tests are given in Table II, p. 282.
The color
changes are designated as yellow, amber, brown, dark brown and
opaque black. It will be noted that the duration of the test varies
directly with the intensity of the heat. Thus at a temperature
of 74O C. or above, the five minute period of boiling, which we
ad.vise, is sufficient to yield the typical black end-reaction in the
presence of reducing sugar, whereas a temperature of 67 C. must
be maintained for a seven minute period and a temperature of
57O C. requires a period of 23 minutes to produce a like effect .
Interfering Substances. A rather large number of substances
have been mentioned by various investigators as interfering with
the Nylander test. Among these may be specially mentioned
album& and various aromatic and wzedicinal substances,such as
rhtlbarb , senna, antipyrin, salol, kairin, turpentine, etc. When
these aromatic and medicinal substances do interfere, and we
believe that this contingency is not of frequent occurrence, it is
highly probable that they interfere indirectly.
It is well known
that the administration of many of the substances mentioned will
cause an increased output of glycuronates in the urine. If this
glycuronate concentration is sufficient a reduction of the bismuth
of the Nylanders reagent will occur. Daiber claims that urine

276

A Study of Nylanders

Reaction

1 Glaw: Deutsche
med. Zeit., xvi, p. 689.
a Btichner
: Mtinch.
med. Woch., xli, p. 99 I
3 Salkowski:
Practicunz:
p. r8r, 1900.
4 Tyson:
Practical
Examination
of Urine,

Tenth

Edition,

p. 91.

Downloaded from http://www.jbc.org/ by guest on January 19, 2016

containing a high indican content will give a positive Nylander

test.
This point was confirmed by Glaw.
Biichne9 reports a
darkening of the reagent when urines rich in uroerythrin
or haematoporphyrin
are under examination, whereas J. Miiller states that
every normal urine upon being strongly
concentrated
through
evaporation
will yield a positive Nylander reaction.
According
to Salkowski3 many very concentrated
sugar-free urines such, for
example, asthose containing chrysophanic
acid, yield a dark color
when subjected to the Nylander test, while Tyson4 has frequently
observed the formation of a peculiar dirty gray precipitate which
he asserts is very confusing.
We have not attempted to verify the claims of the various investigators mentioned.
We have, however, studied the influence
upon the Nylander reaction, of many normal and pathological
urinary constituents.
The first tests of this character we made
were on albumin.
This is universally
considered to possess the
property of darkening the Nylander reagent even when present
in sugar-free urines.
The color formed is a deep amber, cherry or
brown and in the presence of the correct phosphate concentration a characteristic
reddish-brown
color results.
An actual
black color never results, however, when urines containing albumin
are examined and such color as is formed is believed to be due to
the splitting off of unoxidized sulphur from the protein and the
subsequent formation of bismuth sulphide.
In our albumin tests
we first studied the influence of aqueous albumin solutions of
varying strengths
upon Nylanders
reagent.
Typical results are
shown in Table III, p. 283. An examination
of the data here
tabulated will indicate that it is necessary to have a fairly high
albumin concentration
before a color at all similar to that secured
in the presence of sugar is obtained.
Our experiments on testing for sugar in the presence of albumin
demonstrate
a very interesting
fact.
This is to the effect that
albumin when present even in moderate amount in urines containing sugar renders the detection of this sugar by the Nylander
test exceedingly difficult if not totally impossible through the

M. E. Rehfuss and P. B. Hawk

277

1 Bechhold

: LOG. cit.

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formation
of a peculiar colloidal solution.
The data given in
Table IV, p. 284 will demonstrate
this.
This same phenomenon
was noted repeatedly in similar tests.
It will be noted after I or 2
minutes boiling that the tubes containing
albumin possess a
darker tint than do those tubes to which no albumin was added.
However subsequent boiling has no material effect upon this color
whereas in the case of the urines containing sugar the color progressively
deepens until the typical opaque black end-reaction
is reached.
Upon standing 24 hours the tubes containing albumin exhibit no change in color and the formation of no precipitate
while the sugar tubes show an opaque black precipitate
with a
pale yellow supernatant
liquid.
Evidently in the presence of the
albumin some colloidal reaction has taken place such as to prevent the further progress of the true Nylander reaction although
sufficient sugar is present to yield the typical end-reaction in the
absence of albumin.
This phenomenon may be due to the fact,
suggested to us by Professor John Marshall, that a part of the
albumin which has not undergone cleavage holds the bismuth
sulphide in colloidal solution.
Many experiments showed results
similar to those indicated above. Reference to Bechholds
last
table will show a similar condition with the exception that he
used small amounts of serum. We were unable to verify this and
obtained the colloidal solution only when large amounts of albumin and small amounts
of sugar were used.
Larger amounts
of sugar eventually
overcame the tendency toward the formation of the colloidal solution.
Peptone in the presence of Nylanders
reagent yields a color
similar to that obtained in the case of albumin.
(Table III, p.
However ,when we come to test for sugar in the presence
283).
,of these proteins we find a difference..
Whereas in the case of
albumin, as above cited, colloidal solutions resulted instead of the
typical end-reaction
no such phenomenon was observed in the
case of peptone.
(Table IV, p. 284). From what we know of
colloidal solutions this is right in line with what we would expect.
Peptone being dialyzable cannot of course hold the bismuth in a
colloidal state and it therefore precipitates
just as it does in the
absence of protein material.

278

A Study of Nylanders

Reaction

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Further pathological urinary constituents,

other than albumin
and peptone whose influence upon Nylanders
reaction we studied,
included tyrosb, leucin, cholesterol, and cystin. None of these
constituents yielded a reaction simulating the sugar reaction in
the absence of sugar and in the presence of sugar none of them
except tyrosin and leucin exerted any inhibitory influence.
The inhibition produced by tyrosin and leucin was very slight.
The reaction was delayed a trifle but the typical end-reaction
was ultimately obtained.
According to Hammarsten homogentisic acid and alkapton do not yield any reaction resembling
the sugar reaction although the alkapton does cause the formation of a flocculent brownish precipitate.
We could not obtain
any urine containing these substances and hence did not study
their action.
The influence cf excessive amounts of several normal urinary
constituents was studied. Among these were Urea, a&c acid,
There
sodium urate, potassiuwz
urate, creativlivc and phosphates.
was no reduction in any case. This fact emphasizes the advisability of using Nylanders reaction in testing for sugar in urines
which contain a high concentration of uric acid or creatinin inasmuch as it is well known that these substances when present in
the correct amount may cause the drawing of wrong conclusions
when such urines are examined by the copper tests.
Our next tests were made on several acids the greater number of
which are regularly used internally or externally either as such or
in the form of some derivative.
In each instance care was taken
to seethat the alkalinity of the test was not lowered. Table V,
p. 285, gives the results of tests of this character.
The acids
tested included acetic, trichloracetic,
tam&,
boric, tartaric,
pick,
carbolic, arsenious, benzoic, salicylic, chromic, and pryogallic. After
demonstrating that these acids would not of themselves cause a
similar end-reaction to that produced by sugar a seriesof tests were
made with the idea of detecting any inhibitory influence they
might exert u-hen present in urine containing sugar. Data from
these tests are given in Table V, p. 285. An examination of this
table will show that of all the acids studied only three, i. e., trichloracetic, chromic and pyrogallic exerted any inhibitory action
upon the Nylander test. The reaction with pyrogallic acid is the
same (red-brown) no matter whether the urine tested be sugarfree or contain a high concentration of the carbohydrate.

M. E. Rehfuss and P. B. Hawk

279

1 Abt:
Archives of Pediatrics,
xxiv,
p. 275, 1907.
Weitbrecht:
Schweiz.
Wochschr.,
xlvii,
p. 577, 1909.
3 Pfliiger: Lot. cit.
4 Kistermann:
Deutsch.
Arch.
f. k&z.. Med.,
1, p. 423, 189 2.

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The urine from a case in which considerable calcium sulphide

had been administered
was examined but failed to yield a positive Nylander
reaction.
The influence of urotropin
is also of
considerable interest.
This drug yields small quantities of formaldehyde in the urinary tract and according to our experience
this aldehyde reduces Nylanders
reagent.
The claim has been
made by Abt that the urine of patients receiving comparatively
large quantities
of urotropin
gives a pronounced
reduction of
Fehlings solution and has but slight action on the alkaline bismuth tests.
However, Weitbrecht2
has recently reported that
the sugar-free urine of a patient to whom urotropin
was administered
was found to reduce Nylanders
reagent.
This was
attributed
to formaldehyde
resulting from partial decomposition of hexamethylenetetramine
in the organism.
Clilzical
Value
of Nylaulders Reaction.
Pfliiger,3 as before
stated, claims to have secured a positive Nylander reaction when
testing urines known to be sugar-free. He therefore prefers the
copper tests over those of bismuth. Kistermann4 on the other
hand claims that those urines which reduce the Nylander reagent
also reduce the copper tests. As pointed out on p. 273 the communication of Hammarsten seemsto furnish ample demonstration of the inaccuracy of Pfliigers methods. The latter exposes
the test to the heat of t,he water-bath for a period of from fifteen
to thirty minutes. Such procedure cannot fail to demonstrate
at least doubtful if not actually positive reactions with many of
what are claimed to be normal urines.
In Ta.bleVI, p. 286, we have tabulated somedata secured from
prolonged boiling of normal urines on a water-bath in the presence
of Nylanders reagent, according to Pflugers methods. Of the
urines mentioned, Nos. 652-656 represent perfectly normal urines,
and yet the majority of them yielded a positive Nylander test
after sufficient boiling.
The point to be noted however is
that at the end of a five minute boiling period, the period prescribed by us, none of them showed signs of any reducing action.
Numbers 657 and 658 represent morning and evening urine sam-

280

A Study of Nylanders

Reaction

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ples of a person whose urine at this stage was supposed to be perfectly normal and who was subjected to an iridectomypreliminary
Several weeks after the cataract operato the cataract operation.
tion he developed glycosuria which later became fairly well marked.
It will be noted on reference to the table that Fehlings test is
marked doubtful
in the case of No. 658 whereas Nylanders
test yielded a positive reduction
at the end of a half-hour
boiling period.
It is, of course, impossible to determine in this
i,nstance whether the reduction
was due to the presence of a
trace of sugar or was due to the same tendency which causes reduction in normal urines.
Number 659 represents a case which
developed nephritis about one year later.
Our results with Nylanders
reagent, in comparison with other
well known sugar tests, confirm us in our opinion that it is one of
clinical value. In the examination of over seven hundred urines
there were only two instances in which Nylanders
reaction was
positive and Fehlings test negative.
In the first instance there
was a very marked reduction which was assignable to no apparent cause, whereas in the second instance there was a very slight
positive reaction.
We believe with Hammarsten
and Kistermann
that a negative Nylander reaction affords evidence that the urine.
from a clinical standpoint,
may be considered
sugar-free.
The
statement of the latter to the effect that any protein-free
urine
of acid reaction
which gives a negative Nylanders
test may
safely be said to be sugar-free in a clinical sense, seems to be
justified.

M. E. Rehfuss and P. B. Hawk

TABLE

Delicacy

of Nylanders

I
1

amber

amber

deep
amber
deep
amber
deep
amber
deep
amber
amber

2.5

..........

yellow

amber

1.0

..........

amber

0.5

..........

0.25

..........

0.10

..........

pale
yellow
pale
yellow
.V cry pale
yellow
colorless

0.09

..........

colorless

0.08

..........

(colorless

colorless

0.06 ..........

1colorless

0.05 ..........

Gzolorless

0.04..

(zolorless

pale
yellow
ery pal<
yellow
ery palt
yellow
ery pal<
yellow
ery pak
yellow
almost
olorless
olorless

0.03 ..........

(zolorless

olorless

0.02 ..........

(:olorless

olorless

pale
yellow
pale
yellow
pale
yellow
pale
yellow
pale
yellow
pale
yellow
ery pak
yellow
3lorless

(:olorless

olorless

olorless
.___

0.0,.

.........

O.OI..........

........

POSITIVE
OR

amber
yellow

yellow

deep
amber
deep
amber
yellow
brown
yellow
brown
deep
amber
amber
yellow
yellow
yellow
yellow
pale
yellow
pale
rellow
:ry pall
yellow
almost
olorless
olorlesr

JE(1.4TIE
+ or-)

opaque
black
opaque
black
opaque
black
opaque
black
opaque
black
brown
black
brown
black
brown
black
deep
amber
yellow

yellow

yellow

ery pale
yellow
almost
:olorless
:olorless

+
+
+
+
+
+
+
-

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5 ...........

Reagent for Dextrose.

-__

--

281

282

Study of Nylanders
TABLE
InlEuence

Reaction

II
(Centigrade)

of Temperature.

57

--

58
_-

590

70

64

_-

--

72

i4O

--

J
s

Is 7ellow

brown

brown
annbel
P

dark
brown

1mxvll

amber
dark
I 3mwn

a.mber

,*aqu,

black
d

mber

0
I

bl

b
0
t
e

e;
/

E
0
I
XOWD

mxvn

dark

dark

tw3v?n
oPaw
black

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dark
brown

M. E. Rehfuss and P. B. Hawk

TABLE

Influence

of Albumin

2 cc..

. . . .. . . . .

I cc.. . . . . . .
o.gcc . . . . . . . . . . . . .
O.ICC . . . . . . . . . . . . .
0.01 cc . . . . . . . . . . . . .

III

of Peptone
(a)

ALBUMIN.

BOILING

PERIOD

upon

Nylamders

(MINUTES)

amber
yellow
yellow
colorless
colorless

amber
yellow
yellow
colorless
colorless
(b)

amber
brown
amber
brown
yellow
colorless
colorless

Reaction

POBITIVE
OB
NEO*TIB
(+ OR-)

PEPTONE.

POSITIIYE
OR
NEOITIVE
(t
OR-)

2 cc.. . . . . . . . 1
I cc.
0.5 cc..

yellow

. . . . . . Ipale
...........

0. I cc .............
0.01 cc .............

1 amber

yellow/pale
yellow/
very
very
pale yellowpale
yellow
colorless
colorless
colorless
colorless

:zE&
pale

yellow
very
pale yellow
colorless
colorless

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VOLUME OF 2 PER
CENT *LBUMIN
*OIuTION
IN 5 0.0.

and

283

TABLE

Influence

of Albumilz

a%d
(a)

IV

of Peptone

in

amber

deep

cherry

deep cherry
(colloidal)

deep

amber

deep

cherry

deep cherry
(colloidal)

pale

yellow

pale

yellow

amber

5 cc. 2 per cent

peptone
solution + 0.5 cc.
2 per cent dextrose solution.
2 cc. 2 per centi
peptone
solution + 0.5 cc.
2 per cent dextrose solution.
.
E cc. 2 per cent
peptone
solutition + 0.5 cc.
dextrose
solution . . . . . . _ . .
0.5 cc. 2 per cent
peptone
solution
+ 0.5 cc.
dextrose
solution . . . . . . .
0.1 cc. 2 per cent
peptone
solutio
+ 0.5 cc.
2 per cent dextrose solution.
.

amber

deep

amber

opaque
black

black

black

yellow

brown

black

yellow

brown

black

yellow

dark

brown

PEPTONE.

amber

opaque
black

i-

amber

Sugar.

deep

(b)

of

black

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5 cc. 2 per cent

albumin
solu-.
tion
+ 0.5 cc.
2 per cent dextrose solution.
5 cc. 2 per cent
albumin
solution
-t 0.5 cc.
2 per cent dextrose solution.
,
Check test.
5 cc. water
+ 0.5
cc. 2 per cent
dextrosesolution
Check test.
5 cc. water
+ 0.5,
cc. 2 per cent
dextrose
solution1

presence

ALBUMIN

M. E. Rehfuss and P. B. Hawk

TABLE
InfEwnce

285

V
of Acids.

--

Trichloracetic
:.
yellow
Acetic..
. . .. .
yellow
Picric.
. . _ _ . . . . c:herry-red
Tarmic......

yellow

Boric.

.. .. . . .

Tartaric.

....

Carbolic.

....

pale
yellow
pale
yellow
yellow

Chromic..

... .

green

Benzoic..

. . . .:

Arsenious.
Salicylic.
Pyrogallic..

amber
amber
mahog.red
amber

amber
brown
mahog.red
brown

yellow

brown

yellow

brown

amber

brown

Jive

greer

..

yellow

dark
amber
brown

...

yellow

brown

..
-

yellow

amber

--

_-

orange

xanslucen
olive
green

amber
black
mahog.black
brown
black
brown
brown
black
brown
black
t t: ranslucenl
olive
green

brown
brown
black
brown
black
red brown

amber
black
mahog.black
black
brown
black
black
black
ranslucent
olive
green

brown
black
black

black

black

black

I *ed

brown

black

.ed brown

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yellow

yellow

amber
yellow

amber
amber

amber

negative

negative

negative
negative

negative
doubtful

negative

653

654

655
656

651
658

659

yellow

of Prolonged

15

none

gray
wt.
amber
brownblack
brownblack

gray wt.
amber
amber

amber

brownblack
amber,
yellow,

deep

yellow

and

Boiling

VI.

brownish
PPt.
amber
yellow,

amber

yellow

10

in reaction

deep

acid

Influence

were

negative

urines

652

All

TABLE

30

brownblack

amber
black

45

or sugar.

brownblack

gray
wt.
deep brownblack
amber
brown
black
amber
black

PPt.
increased
deep amber,

gray-brown,

albumin

Urines.

amber,

gray
PO.
ieep brownblack
amber
gray-black

deep

PPt.

gray-brown,

__

contained

of Normal

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amber

black

amber
black

gray
wt.
opaque
black
amber
black

deep

brownblack

60

A STUDY OF NYLANDER'S REACTION

M. E. Rehfuss and P. B. Hawk
J. Biol. Chem. 1910, 7:273-286.

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