CHROMOGENIC SUBSTANCES IN URINE. 627 
(a) Indoxyl (indigo-blue and indigo-red). — Indoxyl (cf. pp. 607 
and 631) easily oxidises 1" indigo-blue, or to the isomeric substance 
indigo-red. The relation between indoxyl and Its lilue derivative is 
expressed by the following equation:— 
C 6 H 4 / C <£E)%CH + 2 = 0^)0 = 0(^)^ + 2^0 
(indoxyl) (indigo-blue) 
The formula of indigo-red is C H 4 ' ^tt /C = C<^ a „ \N, and it 
arises, like its blue isomer, when, by oxidation, four atoms of hydrogen 
are removed from two molecules of indoxyl. Oxidising reagents when 
added to urine may, according to the conditions of the experiment, give 
rise to the formation of either or both of these coloured derivatives. 
The blue substance, however, is more easily and more generally 
obtained. 
It is of great rarity for the urine to be actually pigmented 
with indigo-blue. As we have already seen (p. 607), the urinary 
indoxyl is excreted in the form of a conjugated sulphate, and 
this compound resists oxidation. Only when the indoxyl is first 
liberated from its combination does the action of oxidising reagents 
produce the blue 1 colour. It is stated, however, that the urine of 
cholera may sometimes exhibit a blue shade from the presence of 
indigo-blue. "We have seen that the amount of indoxyl sulphate is 
increased in the urine whenever bacterial putrefaction of albuminous 
substances is occurring to a greater extent than usual, whether in the 
bowel or elsewhere in the body (putrid abscesses, etc.). The most 
ready method of demonstrating the amount of indoxyl is by converting 
it into indigo-blue. Jaffa's test. 1 — The urine is mixed with an equal 
bulk of strong hydrochloric acid, by which means the " indican " 
(indoxyl sulphate) is decomposed and the indoxyl liberated. With a 
pipette, a solution of a hypochlorite is now added to the mixture drop 
by drop, when, by oxidation of the indoxyl, indigo-blue is formed. By 
shaking up the liquid with chloroform, a solution of the blue substance 
is obtained in the latter (Stockvis, Senator, and others). Otherwise, 
a crystal of potassium chlorate is placed at the bottom of a test tube 
and covered with the urine to be examined. Strong hydrochloric acid 
is then allowed to run down the side of the tube so as to reach the 
crystal without mixing -with the urine. The latter floats upon the acid, 
and at the junction of the fluids a blue ring is seen of intensity varying 
with the amount of indoxyl present. 
But indigo-blue is itself an easily oxidisable substance. It is 
instantly decolorised by nitric acid, and without difficulty by hypo- 
chlorites. In Jaffe's test, as above described, it is therefore necessary 
to add the oxidising agent with great care, or the blue colour will 
disappear as soon as formed. In Obermayer's 2 method, the urine is 
first precipitated by acetate of lead, and filtered : to the filtrate is added 
an equal bulk of strong hydrochloric acid, containing two or three parts 
per thousand of ferric chloride. The mixture is shaken for a short 
time, and the liberated pigment taken up, as before, in chloroform. In 
1 Arch.f. d. r/es. Physiol., Bonn, 1870, Bd. iii. S. US. 
- Wicn. Id in. Wchnschr., 1S90, S. 176. 
