A SIMPLE METHOD FOR DETECTING AND ESTIMATING 1NDICAN IN THE URINE BY MEANS OP THE COTTON- WOOL PLUG TEST.*

By S. N. GORE. i,.M.S. (Bombay), Bombay Bacteriological Laboratory.

Indican is derived from indol, and unlike the other ethereal sulphates which are derived in part from metabolism, originates practically wholly from putrefactive processes. It, alone, therefore, and not the total ethereal sulphates, has been taken as an index of such putrefac- tion (1). The determination of indican depends upon

its decomposition and oxidation of the liberated indoxyl into indigo-blue ; and of the methods based on this principle, Obermeyer's test is in

general use. This test is carried out by mix- ing an aliquot portion of urine with an equal volume of Obermeyer's reagent,f and shaking the mixture with a few c.c. of chloroform which on standing sinks to the bottom and becomes blue. Normal urines?i.e., those con-

taining traces of indican?impart a very faint blue to the chloroform, while in marked indi- canuria the chloroform becomes deeply blue. The depth of the colour is proportional to the amount of indican contained in a sample of urine, and this fact is often made use of for an approximate estimation of the amount of indican by using the colour of Fehling's solu- tion as a standard(2). Obermeyer's test, though fairly simple in

execution, does not meet all the requirements of a routine test, and until such a test is avail- able, indican may continue to be tested

irregularly as at present, and probably so long will its true significance, if any, remain un- determined. It was, therefore, interesting to find that during an investigation regarding the scope of the applicability of the cotton-wool plug test(3)?primarily devised by the author for applying Ehrlich's reaction to the sputum to a variety of other materials,?urine was found to respond positively to this test; and in view of the fact that the technique of this test, compared with that of Obermeyer's, is simple,

* Being a paper read at the Medical Research Sec- tion of the Indian Science Congress, 1924.

t Strong hydrochloric acid containing 0.2 per cent, of ferric chloride,

394 THE INDIAN MEDICAL GAZETTE. [Aug., 1924.

delicate and economical both in the time and the reagents required to carry it out, experi- ments were instituted to find out the extent to which the cotton-wool plug test could

replace Obermever's for the determination of indican in urine. The first point that required elucidation in

such an inquiry was to ascertain whether the results obtained by these two tests corre-

sponded qualitatively, and for this comparative study the following procedures were adopted for their application to urine.

Cotton-xvool plug test.?2 c.c. of urine was

put in a small test tube (5 in. by f in.) which was fitted with a plug made of white absorbent cotton-wool. The plug was removed, its under surface moistened with a drop or two of the potassium persulphate and the p-dimethylbenz- aldehyde solutions,* and replaced. The urine was then boiled intermittently for about a

minute.

Obcrmeycr's test.?2 c.c. of urine was put in a small test tube (5 in. by J in.). 2 c.c. of Obermeyer's reagent added, well mixed, and the mixture allowed to stand for fifteen minutes. At the end of this period 2 c.c. of chloroform was added to the mixture, the mouth of the tube closed with the thumb, and the liquids gently inverted ten times. Under these standard conditions a number

of samples of urine were examined and it was found (/) that there was not a single instance where the cotton-wool plug test failed to res- pond positively when indican was detected by Obermeyer's test, and (ii) that the intensity of the pink colour on the cotton-wool appeared more or less proportional to the depth of the blue colour in the chloroform.

In view of these results, the next question was to decide upon a procedure by means of which the second finding could be verified and the cotton-wool plug test applied for quanti- tative purposes. In the absence of quantita- tive methods suitable for routine work, a few

preliminary experiments showed that dilution tests would best meet the requirements of such a comparative study.

Accordingly ten old samples of urine were secured, and four successive half-dilutions were made from each sample. Two c.c. of the urine and of the four dilutions were put in five test tubes, and two such sets prepared for each sample. One set was examined by means of the cotton-wool plug test and the other by Obermeyer's test. The results of these tests are ?fiven in Table I.

Tablk I.

Showing the results of the dilution tests.

No. of sample of urine.

Un- diluted urine.

. H O

1

2

3

4

5

6

7

8

9

10

+

<L> <V

o1*

i dilu- tion.

pl,

'Si

> O <L> 0/

+ +

?f i +?

+ I 4-

i dilu- tion.

oH,o u tn QJ <U

i dilu- tion.

CA

<U

O

U. IA <u <u XJC-I o

tV dilu- tion.

d<

a

Vfc/ <-

o

From this table it will be seen:?

(1) That all samples of urine responded positively to both the tests.

(2) That the highest dilutions which so

responded were found to correspond, though different samples did so in different dilutions as follows:?

Nos. 7 and 9 showed positive results in the undiluted samples only.

Nos. 1, 4, 5 and 8 showed positive results up to j dilutions of the samples.

Nos. 2, 3 and 10 showed positive results up to 4 dilutions of the samples.

No. 6 showed positive results up to ? dilu- tions of the sample. The above results thus confirm the findings

of the first series of experiments in the fact that urine contains a volatile chromogen which, as determined by the cotton-wool plug test, is

proportionate to the indican as determined by Obermeyer's test. The question regarding the nature of this volatile chromogen and how it is related to indican is under investigation and will be dealt with in a future communication. However, in view of the fact that this chromo- gen resembles indol in its response to Ehrlich's reaction in the cotton-wool plug test and has been found to be colorimetrically proportionate to the indican, an attempt has been m^de to ascertain whether the results of the dilution tests could be expressed in terms of the indol- equivalent of the indican. As the result of a comparative study with

nine different methods of applying Salkowski's '

nitroso-indol' and Ehrlich's '

rose-indol' re-

actions for the detection of indol in bacterial

* Potassium persulphate 1 grm. Distilled water 100 c.c.

P-dimethylamido-benzalde- liyde 1 grin., Absolute alcohol 95 c.c., Concen- trated hydrochloric acid 20 c.c.

i\otc.?These two solutions are conveniently stocked in glass-stoppered bottles (Pattern T.K.).

Aug., 1924.] DETECTING AND ESTIMATING INDICAN TN URINE: GORE. 395

cultures, M alone and Gore found that the

cotton-wool plug" test best fulfilled the condi- tions of a routine test, and detected indol in as small concentrations as 0.0005 mg. per c.c.

of liquid(4). Accordingly, a few experiments were performed on 2 c.c. quantities of liquids containing varying concentrations of indol, with the result that liquids containing on an average about 0.0005 mg. of indol per c.c. were found to impart a faint but distinct pink to the cotton-wool, while liquids having lower con- centrations failed to show any pink colouration. 0.0005 mg. of indol per c.c. was, therefore, taken as the minimum concentration in which indol could be detected by the cotton-wool plug- test, and in order to express the results of the dilution tests in terms of mg. of indol equi- valent per c.c. of the urine, one had only to

multiply the strength of the highest positive dilution by 0.0005, as shewn below.

If a sample of urine responded positively to the cotton-wool plug test only in the undiluted

Table: II. Showing the details of an experiment in which the '

indol equivalent '

of the indican present in the urine was estimated by means of the Cotton-Wool Plug Test, directly from the urine, and indirectly, from its distillate and residue.

Urine

1st distillate

2nd ,,

3rd

4th

5th

Residual from the distillate

Results of Cotton-Wool Pluo Test applied to

3.^ rs 3

cr a vr.

P

+

+

+

+

+

+

0

Mg. indol-equivalent per c.c. i of the undiluted liquid ob

Half dilutions of the liquids, j rained by multiplying the strength of the highest dilu-

+

+

+

+

+

0

0

+

+

+

+

0

0

0

0

+

+

0

0

0

+

+

0

0

0 I 0

tion by "0005.

Mg. of indol- equivalent pe c.c. of urine.

0

0

0

0

0

0

4 x *0005 = '002

16 x -0005 = '008

16 x -0005 - -008

4 x -0005 = '002

2 x '0005 = '001

1 x '0005 = '0005

0

?002

'00195

state the indican-content ot the urine would be expressed as 0.0005 nig. of indol-equivalent per c.c.

If a sample of urine responded positively to the cotton-wool plug" test in its ^ or \ or -J dilu- tion, the indican-content of the urine would be expressed as 2 X 0.0005 = 0.001 mg. or

4 X 0.0005 = 0.002, or 8 X 0.0005 = 0.004 mg. of indol-equivalent respectively per c.c.

With the object of ascertaining whether the above procedure of estimating the volatile

chromogen of urine by means of the cotton- wool plug test and expressing the results in terms of indol was capable of yielding approxi- mately correct results, a few experiments were instituted in each of which 100 c.c. of urine was divided into two equal portions. One

portion was directly examined by the above procedure and the results thereof calculated in terms of indol. The other portion was sub- jected to distillation and 25 c.c. of distillate obtained in five lots of 5 c.c. each. Each of these 5 c.c. distillates was examined by the above procedure, the results thereof calculated in terms of indol, and added up. The residual urine left in the flask was also examined but

gave a negative result. These experiments showed that the error between the indol-

equivalent estimated directly from the urine and from its distillate did not exceed 5 per cent. The details of one of these experiments are given in Table II from which it will be seen that the indol concentration of the urine as

estimated directly from the urine was 0.002 mg. per c.c., while that from its distillate was 0.00195 mg". per c.c. On the basis of all the above results, the

following procedures have been devised for the detection and the approximate estimation

of indican in urine. Hie methods ot procedure are as follows :?

Detection of Indican by the Cotton-Wool Plug T est.

1. Put 2 c.c. of urine into a small test tube

(5 in. by f in.) and fit it with a plug made of white absorbent cotton-wool.

2. Remove the plug, moisten its under-surface with a drop or two, first of the potassium per- sulphate solution and then of the p-dimethyl- benzaldehyde solution, and replace it.

3. Bring- the urine to the boil by intermit- tent heating in a low flame and if the urine contains indican, half a minute's or a minute's further such boiling, imparts a pink colour to the moistened under-surface of the plug. This colouration varies from a faint but distinct

396 THE INDIAN MEDICAL GAZETTE. [Aug., 1924.

pink to a deep pink according to the indican- content of the urine. In the case of a nega- tive result, i.e.. absence of indican, no pink colour is seen on the cotton-wool.

Estimation of Indican by the Cotton-Wool

Plug Test. Having ascertained that a sample of urine

has responded positively to the cotton-wool

plug test as described above, estimation of

indican is effected by applying the above described cotton-wool plug test to one or

several of a series of successive half dilutions of the urine as follows:?

1. Measure 2 c.c. of urine into a 5 c.c.

graduated cylinder, dilute it with water to

4 c.c. and mix well. From this half-dilution of

urine pour out 2 c.c. into a test tube gnd test as above. If the test is negative, no further dilutions are made.

2. If the half-dilution gave a positive result, dilute the remaining 2 c.c. of the half-dilution in the measure glass to 4 c.c. and mix well. From the resulting quarter-dilution pour out 2 c.c. into a test tube and test as above. If

the test is negative no further dilutions are

made. 3. If the quarter-dilution gave a positive

result, dilute its remainder to 4 c.c. and mix

well. From the resulting eighth-dilution pour out 2 c.c. into a test tube and test as above.

In the case of a negative result, no further dilutions are made, whilst in the case of a

positive result further dilutions are similarly made and tested. In fact, successive half-

dilutions are required to be made until a posi- tive dilution is succeeded by a negative one, and it is this positive dilution, which is taken

as the highest dilution in which the urine is

said to respond positively to the cotton-wool plug test. The quantitative results thus obtained may

be expressed either (/) in terms of the

strength of the highest dilution in which the urine responded positively to the cotton-wool plug test; or (//) in terms of mg. of the indol-

equivalent of indican per c.c. of the urine; this is effected by multiplying the strength of the highest positive dilution by 0.0005 as explained in the body of the paper. For example, in case a sample reacted positively in ? as its

highest dilution, the indol-equivalent of the indican present in the urine would be 8 X 0.0005 = .004 mg. per c.c. of the urine.

Thus not only is the presence of indican in urine detected but an approximate estimate of its amount obtained as well, more simply, rapidly and economically by determining the volatile chromogen in the urine by means of the cotton-wool plug test than by Ober- meyer's test. Further, the cotton-wool plug- test possesses an additional advantage in the fact that its results are not liable to be vitiated by the presence of iodine in the urine which imparts a pink colour to the chloroform in

Obermeyer's test and is only got rid of by further treatment of the urine with sodium

sulphite. ATotc.?One point to he remembered while

applying the cotton-wool plug test to urine is that a volatile product is being tested for, and it is,, therefore, essential that the sample of urine should be examined within a few hours of its being voided.

Sum mary. 1. The detection of indican in urine is, as

a rule, effected by its decomposition and oxi- dation of the indoxyl set free to indigo-blue ;

and Obermeyer's test, which is based on this

principle, is the one in general clinical use

chiefly for the purpose of detecting and occa- sionally for the rough estimation of the amount of indican present in urine.

2. Obermeyer's test, though simple, does not meet all the requirements of a routine

test, and consequently fails to be used routine-

ly in clinical work. In this paper it has been shown that the cotton-wool plug technique devised by the author primarily for apply- ing Ehrlich's reaction to detect indol in

sputum, could also be used to determine indi- can in urine ; and compared with Obermeyer's test has been found to be more simple, rapid and economical in the reagents required, besides, being independent of the drugs taken by the patient.

3. Further, since the volatile chromogen in

the urine responding positively to the cotton- wool plug test resembles indol in its reaction and is proportional to the indican as deter- mined by Obermeyer's test, a quantitative method based on dilution tests has been devised

whereby the amount of indican present in urine can be approximately estimated and expressed in terms of its indol-equivalent.

References. 1. Todd, J. C.?Clinical Diagnosis, 4th edition, p. 133. 2. Follin Otto.?Laboratory Manual of Biological

Chemistry, p. 105. 3. Gore, S. N.?Ind. Jour. Med. Research, VIII, 3.

p. 505. 4. Malone, R. H.. and Gore, S. N.?Ibid., p. 490.