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0307-4412(94)00084-0
Test for Occult Blood.
The Benzidine Reaction: a Text Book Error
USHA ANAND, RAJNI AGARWAL and C V ANAND
Department o f Biochemistry
M S R Medical College
Bangalore 560 054, India
Introduction
The presence of blood/haemoglobin in the urine, both haema-
turia and haemoglobinuria, is usually indicative of a pathological
condition. It can be detected using ready-made dipsticks or by
the simple benzidine test. Most of the text-books in clinical
chemistry give a brief account of the manner in which this test
can be carried out and also the principle on which it is based. H °
In this test the haem moiety of haemoglobin has a 'peroxidase-
like' activity which brings about the decomposition of hydrogen
peroxide to liberate nascent oxygen. This oxygen is allowed to
react with a chromagen such as benzidine or o-tolidine which
undergoes oxidation to produce a blue or a green coloured
product ]CAUTION: SEE BELOW]. The appearance of a
transient blue or a green colour is indicative of a positive test for
blood or haemoglobin. The intensity of the final colour has been
reported to be proportional to the amount of haemoglobin
present in the urine. 7'9't° It is also considered to be a very
sensitive test and can detect the presence of haemoglobin at
concentrations as low as 2 mg/dl of urine or if blood is present,
erythrocytes at a level of 10/1-1 of urineJ '2"7`s't°'12 The equation
for the principle of the method is usually represented as follows
haem
H2Oz + Chromogen > oxidized + H20
peroxidase chromogen
activity (coloured)
Catalase and Peroxidase
Erythrocytes are known to contain several enzymes and some of
them are capable of acting upon HzO2.11 Most relevant among
them is catalase (H2Oz'H202 oxidoreductase EC 1.11.1.6) which
can bring about the rapid decomposition of H202 using one
molecule as an oxidant and another as reductant:
2H202 catalase 2H20 + 02
In addition, catalase also exhibits peroxidase activity and
catalyses the oxidation of various hydrogen donors in the
presence of relatively lower concentrations, of H202. I 1 Catalase
levels in erythrocytes are high eg about 15 x 104 IU/g of
haemoglobin.l~ This enzyme could also bring about the reaction
which has been attributed solely to the peroxidase activity of
haem of haemoglobin. Several books describing this particular
test do not mention the possible contribution of catalase, and in
fact the peroxidative activity of this enzyme is several-fold higher
than that of the haem portion of haemoglobin.
Although the catalase of erythrocytes has been ignored, some
text books 3'5'9"1° mention interference by the perioxidase activity
of pus cells and of bacteria which could lead to a false positive
test for blood. In order to eliminate this interference it is
recommended that if pus cells have been detected by micro-
scopy, the urine should be boiled so that the enzyme is rendered
inactive. The same argument holds good for erythrocyte
catalase. However, most of the procedures, using either strips
impregnated with an organic peroxide and a chromogen or
procedures using reagents and test tubes, do not advocate prior
boiling as a routine measure.
BIOCHEMICAL EDUCATION 22(4) 1994
Simple test
In order to prove that catalase indeed contributes to the positive
test for blood, a simple test was carried out. Aiiquots of urine
samples known to contain occult blood (and not to contain pus
cells) were treated with H20 z. There was a brisk effervescence
with copious evolution of oxygen. The entire test tube was filled
with a thick froth. Another aliquot of the same sample was
boiled for 1-2 minutes and treated with H202. The effer-
vescence was very scanty proving the existence of a heat-labile
factor capable of decomposing HzO2. When treated with
benzidine in glacial acetic acid both gave a dark blue/green
colour. There was no visual difference in the rate of colour
development or in its intensity. The colour development step, ie
the oxidation of benzidine, is extremely sensitive and therefore
cannot be used to show any difference in the quantity of H202
decomposed in the two samples, the fresh urine sample in which
both catalase and haem are present and the second in which the
catalase activity has been destroyed and only the pseudo-
peroxidase activity of hemoglobin remains. The difference in the
effervescence when treated with H202 was however significant
and indicated that the catalase activity was several-fold higher
than that due to haem of haemoglobin. Although catalase is also
a haem-protein the amount of haem present in this form is
negligible when compared to the haem of haemoglobin.
Carcinogens
A significant point is that benzidine and o-tolidine are potent
carcinogens: skin absorption or inhalation of the powder may
lead to bladder cancerJ ° In many countries restrictions have
been imposed in the manufacture and distribution of these
compounds although in some developing countries the use of
these chemical continues. Care in handling these chemicals is
important and alternatives should be sought.
Varley's Practical Clinical Biochemistry 12 gives a brief
description of three different procedures for the detection of
blood in faeces. Chromogens that have been suggested are
diphenylamine in combination with N,N-di(2-hydroxyethyl)-
glycine, 2,2-azino di-(3-ethylbenzthiozoline sulphonic acid) or
ABTS, and chlorpromazine-EDTA. These could be adapted for
the detection of blood in body fluids, as basically the reaction is
the same.
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