j. biol. chem.-1910-rehfuss-273-86

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A STUDY OF NYLANDERS REACTION.

BY M. E. REHFUSS AND P. B. HAWK.

(From the Laboratories of Ph ysio log ical Chem istry of the Department of Medi-

cine of the University of Pennsylvania and the University of Illinois.)

(Received 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 reac-

tion; (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 examina-

tion 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 Pfli iger 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 Bechh old: Zeitschr. f. physiol. Chem., xlvi, 06.

. 370, 19

2 Rehfuss and Hawk: Th is Joural, vii, 267,. 1910.

3 Pfltiger: Arch. f. d. Ph ysiol., cxvi, 265 andes.

pp.

533, 1907.

4 Ham marsten: Ibid., cxvi,

p. 517, 1907.

273

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274

A Study of Nylanders Reaction

to give a positive reaction when subjected to Pfliigers manipu-

lation. Pfliiger, in criticising Hammarstens procedure says that

heating over a free flame cannot fai l 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 moreuni-

form heat is secured through his practice of using the water-

bath.

In his tests upon the inhibitory action of mercury and of chloro-

form 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 Pfli iger, Hammarsten, and Bechhold

we are forced to the conclusion that the five minute boi ling 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 bot-

tom of which forms an admirable background for observing and

comparing the various color changes. The constancy of condi-

tion, the fact that several tests may be made simultaneously,

together with the very material shortening of time seem to us suffi-

cient 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 boi ling

water-bath for five minutes. The tube is then removed and ex-

amined before a white background. A black end-reaction indi-

cates the presence of reducing sugar in the urine under examina-

tion.

The Delicacy

of

the Test.

Inasmuch as there is such great

diversity of opinion as to t.he delicacy of Nylanders test we con-

sidered it worth while to investigate this point.

Certain text-

books state that the test will detect sugar when present in a con-

centration of

0.025

per cent whereas other books set values of

0.05 per cent and

0.1

per cent as he 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

1 We used the following formula for making the reagent : z grams bismuth

subnitrate; 4 grams Roch elle salt; IOO cc. IO per cent potassium hydroxide.

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M. E. Rehfuss and P. B. Hawk

275

being recorded in Table I, p.

281.

Inasmuch as a positive reac-

tion 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

sugar i. e., Moores reaction.

Solutions of lzevulose, maltose

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 end-

reaction 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

1 Daiber: Corresp. Schwe izer

Aerzte, xxiv, p.

38.

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276


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 haema-

toporphyrin 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 in-

vestigators 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 concentra-

tion 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 al l 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 con-

taining sugar renders the detection of this sugar by the Nylander

test exceedingly difficult if not totally impossible through the

1 Glaw: Deu tsche med . Zeit., xvi, p. 689.

a Btichner : Mtinch. med. Woch ., xli, p. 99 I

3 Salkowski: Practicunz:

p. r8r,

1900.

4 Tyson: Practica l Examination of Urine, T enth E dition, p. 91.

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277

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 pro-

gressively deepens until the typical opaque black end-reaction

is reached. Upon standing

24

hours the tubes containing albu-

min 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 pre-

vent 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 albu-

min and small amounts of sugar were used. Larger amounts

of sugar eventually overcame the tendency toward the forma-

tion 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.

283).

However ,when we come to test for sugar in the presence

,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.

1 Bechh old : LOG. cit.

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278


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 homo-

gentisic acid and alkapton do not yield any reaction resembling

the sugar reaction although the alkapton does cause the forma-

tion 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,

sodium urate, potassiuwz urate, creativlivc and phosphates.

There

was no reduction in any case. This fact emphasizes the advisa-

bility of using Nylanders reaction in testing for sugar in urines

which contain a high concentration of uric acid or creatinin inas-

much 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 see hat 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., trich-

loracetic, 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 sugar-

free or contain a high concentration of the carbohydrate.

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279

The urine from a case in which considerable calcium sulphide

had been administered was examined but failed to yield a posi-

tive Nylander reaction. The influence of urotropin is also of

considerable interest. This drug yields small quantities of form-

aldehyde 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 bis-

muth tests. However, Weitbrecht2 has recently reported that

the sugar-free urine of a patient to whom urotropin was admin-

istered was found to reduce Nylanders reagent. This was

attributed to formaldehyde resulting from partial decomposi-

tion 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 com-

munication of Hammarsten seems o furnish ample demonstra-

tion 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 pre-

scribed by us, none of them showed signs of any reducing action.

Numbers 657 and 658 represent morning and evening urine sam-

1 Abt: Archives of Pe diatric s, xxiv, p. 275, 1907.

Weitbrecht: Schweiz. Wochschr., xlvii,

p. 577, 1909.

3 Pfliiger: Lot.

cit.

4 Kisterm ann: Deu tsch. Arch. f. k&z.. Med., 1, p. 423, 189 2.

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280 A Study of Nylanders Reaction

ples of a person whose urine at this stage was supposed to be per-

fectly normal and who was subjected to an iridectomypreliminary

to the cataract operation.

Several weeks after the cataract opera-

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 re-

duction 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 appar-

ent 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.

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TABLE I

Delicacy of Nylanders Reagent for Dextrose.

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

2.5 ..........

1.0 ..........

0.5 ..........

0.25 ..........

0.10 ..........

0.09 ..........

0.08 ..........

0.0,. .........

0.06 ..........

0.05 ..........

0.04.. ........

0.03 ..........

0.02 ..........

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

-

I

--

.V

.

. (

.

. 1

. G

. (

. (

. (

. (

-

t

amber

yellow

pale

yellow

pale

yellow

cry pale

yellow

colorless

colorless

colorless

colorless

colorless

zolorless

zolorless

zolorless

:olorless

:olorless

1

amber

amber

amber

amber

yellow

pale

yellow

ery pal

j. biol. chem.-1910-rehfuss-273-86

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