2007, Vol 2 No 2, Article 16
Review of a Practical
Electrometric method
for determination of Blood
and Tissue
Cholinesterase activities in
Animals
Fouad. K. Mohammad
Department of Physiology, Biochemistry and Pharmacology, College of
Veterinary Medicine,
University of Mosul, P. O. BOX 11136,
Mosul, Iraq
(E-mail: [email protected])
SUMMARY
Measurement of cholinesterase activity is
of diagnostic value in cases of poisoning with organophosphate and
carbamate insecticides. The enzyme is inhibited to various extents with
concomitant appearance of signs of cholinergic hyperstimulation. The
present report introduces and reviews a practical and simple
electrometric technique to measure blood (plasma, erythrocyte and whole
blood) or tissue (brain, liver and muscle) cholinesterase activities in
animals as well as to measure blood cholinesterase activities in man.
Typically, the procedure involves the
addition of 0.2 ml of blood sample or tissue homogenate to 3 ml of
distilled water followed by 3 ml of barbital-phosphate buffer solution
(pH 8.1). The pH (pH1) of the mixture is measured, and then 0.1 ml of
7.1% of acetylcholine iodide or 7.5% acetylthiocholine iodide, as a
substrate, is added. The reaction mixture is incubated at 37º C for
20-40 minutes according to the animal species. The pH (pH2) of the
reaction mixture is measured after the end of the incubation period. The
unit of enzyme activity is expressed as Δ pH / incubation time= pH1- pH2
- (Δ pH of the blank). The blank is without the enzyme source.
Literature are cited regarding the expected normal cholinesterase
activities in man and several animal species including mice, rats,
sheep, goats, cattle, chickens, fish and wild birds. The method was
found to be efficient, simple, accurate and reproducible for possible
monitoring of exposure of man or animals to organophosphate or carbamate
insecticides.
KEY WORDS
Carbamate,
Cholinesterase, Electrometric method, Insecticide, Organophosphate
INTRODUCTION
Organophosphates and carbamates are widely
used insecticides in veterinary medicine, public health and in agriculture
(1-3). As a result man and animals are at risk of exposure to these
insecticides. The single most important toxic action of organophosphate
and carbamate insecticides is inhibition of acetylcholinesterase activity
leading to accumulation of acetylcholine at the nerve endings which in
turn produces signs of poisoning characterized by nicotinic, muscarinic
and central nervous system effects (1,4,5).
Measurement of blood (plasma or erythrocyte) and tissue cholinesterase
activities is a useful tool for monitoring exposure to organophosphate and
carbamate insecticides and diagnosing their poisoning (3,6-8). Usually a
20-30% decrease in serum cholinesterase activity suggests exposure to
anticholinesterases (9). More than 50% inhibition of cholinesterase
activity supports the diagnosis of poisoning and indicates a hazardous
condition (6-9). The aim of the present review was to introduce a modified
electrometric method advocated for measuring blood or tissue
cholinesterase activities in various animal species and to present normal
enzyme activity as reported in the literature by our research group.
METHODS FOR
MEASURING CHOLINESTERASE ACTIVITY
Various
colorimetric and electrometric (potentiometric) methods are
available for the determination of cholinesterase activity
(6,8,10-14). One of the principle methods for measuring blood
cholinesterase activity is the electrometric method which is based
on the hydrolysis of acetylcholine and production of acetic acid
which in turn decreases the pH of the reaction mixture (6,10,12).
The original electrometric method of Michel (15) is commonly used in
man (6). However, the method is not efficiently applicable to
measure cholinesterase activities of different animal species
(6,10,11). This is because of the inherent variations in blood or
tissue cholinesterase activities between different animal species
(6,11,16-18) and the special need for different buffer compositions,
reaction temperatures, incubation times and sample volumes
(10,19-21). In addition, the original electrometric method cannot be
recommended for detection of cholinesterase inhibition induced by
carbamates (10,22,23). Carbamylated cholinesterase is unstable in
the reaction mixture of the electrometric method of Michel because
of considerable sample dilution and long incubation time (60 min)
(10,22,24).
ELECTROMETRIC
CHOLINESTERASE DETERMINATION
Various
modifications of the electrometric method are available for
measuring blood cholinesterase activity in animals
(6,10,11,13,14,20,25). These modifications include increasing sample
volume, increasing or decreasing incubation time, increasing
incubation temperature or using buffers of different compositions
(6,10,11). One simple modification of the electrometric method is
that of Mohammad et al. (21) which was introduced for rapid
measurement of erythrocyte and plasma cholinesterase activities in
sheep. The method was then applied successfully on several animal
species such as mice (26,27), rats (28-30), goats (31,32), chickens
(33-35), wild birds (36) as well as man (27,37,38). More recently,
normal reference values for plasma, erythrocyte or whole blood
cholinesterase activities as determined by the described
electrometric method were reported in man (27,38) as well as in
sheep, goats and cattle (39-41).
Table 1 shows the reported normal blood (plasma, erythrocyte or
whole blood) and tissue cholinesterase values in man and different
animal species as measured by the presently described method. These
cholinesterase values could be starting reference points for future
studies applying the presently described electrometric method in
biomonitoring of exposure of man and animals to anticholinesterase
insecticides. The method is characterized by its simplicity,
reproducibility, accuracy and one-step short incubation time (20-40
minutes) depending on the animal species (Table 1). The coefficient
of variation of the method is usually low (<10%) (21,27,32). It can
be applied on several samples within a relatively short period of
time in comparison with the original Michel method. Further, the
present method, in contrast to the original electrometric method of
Michel (15), can detect cholinesterase inhibition induced by
carbamate insecticides such as carbaryl (34,35) and methomyl (28).
The method correlates well with the electrometric method of Michel
(21,37) and with the colorimetric method of Ellman (34,37,43) in
measuring cholinesterase activity. The method also substantially
decreases handling of the reaction mixture (e.g., preliminary 10 min
incubation time) which is found in other electrometric methods
(6,10). In contrast to other electrometric methods, only one type of
buffer (barbital-phosphate) solution is used in the present method
for the biological samples (21).ay be made by
blood examination during the acute stage.
PROCEDURE FOR
ELECTROMETRIC MEASUREMENT OF BLOOD CHOLINESTERASE ACTIVITY
Venous blood samples are collected using heparinized test tubes
(44). Plasma is separated from erythrocytes by centrifugation at
3000 rpm for 15 minutes. Figure 1 outlines the steps for measuring
plasma, erythrocyte or whole blood cholinesterase activities. The
reaction mixture in a 10-ml beaker contains 3 ml distilled water,
0.2 ml plasma, erythrocytes or whole blood and 3 ml of
barbital-phosphate buffer solution (pH 8.1) (21). The pH of the
mixture (pH1) is measured with a glass electrode using a pH meter,
then 0.10 ml of 7.1% aqueous solution of acetylcholine iodide or
7.5% acetylthiocholine iodide is added to the mixture which is
incubated at 37º C for 30 minutes. The incubation period in man and
cattle is 20 minutes, 30 minutes in sheep, rodents and avian species
and 40 minutes in goats (Table 1). At the end of the incubation
period, the pH of the reaction mixture (pH2) is measured. The enzyme
activity is calculated as follows:
Cholinesterase activity (∆ pH/incubation time) = (pH1 – pH2) - ∆ pH
of blank
The blank is without the blood sample. The unit of cholinesterase
activity is expressed as Δ pH/incubation time, e.g. ∆ pH/30 minutes.
The barbital-phosphate buffer solution (pH 8.1) consists of 1.237 g
sodium barbital, 0.63 g potassium dihydrogen phosphate and 35.07 g
sodium chloride dissolved in one liter of distilled water (21). The
pH of the buffer is adjusted to 8.1 with 0.1N hydrochloric acid. This buffer
solution is suitable for both blood and tissue samples (21,26). For
comparison purpose it is possible to use a single incubation period
of 30 minutes for sheep, goats and cattle (41).
PROCEDURE FOR
MEASURING TISSUE CHOLINESTERASE ACTIVITY
Samples (0.5-1 g)
of brain, liver or muscle are homogenized in the barbital-phosphate
buffer solution (pH 8.1) at 100 mg wet tissue weight/3 ml with a
teflon homogenizer using 25% of the maximum velocity of the electric
homogenizer (26,29,34). Glass homogenizer can also be used for
manual homogenization. Homogenization is performed on an ice bath,
and all tissue homogenates are kept on ice before cholinesterase
determination. For tissue cholinesterase activity, 0.2 ml of the
tissue homogenate is used instead of the blood aliquot in the
reaction mixture described above. The rest of the procedure is the
same as in the case of the blood (Figure 2).
MONITORING
EXPOSURE TO ORGANOPHOSPHATE OR CARBAMATE INSECTICIDES
The described
electrometric method was reported to be efficient in detecting in
vitro or in vivo cholinesterase inhibition in the blood or tissues
of man and different animal species (21,27,28,29,34,35,38,45).
Veterinarians and agriculture workers exposed to insecticides during
their routine work had relatively low plasma and erythrocyte
cholinesterase activities as detected by the present electrometric
method (27,45). The method was also utilized to detect the extent of
blood or tissue cholinesterase inhibition in animals intoxicated
experimentally with organophosphate insecticides such as dichlorvos
(27,29,34,35), diazinon (31,43,46) and malathion (33,45) as well as
with carbamate insecticides such as carbaryl (27,34,35) and methomyl
(28). The method was also applied to detect plasma or tissue
cholinesterase inhibition in wild birds (rock dove, quail, sand
grouse and starling) (43) and chickens (33-35) intoxicated with
organophosphates and carbamates and in fish (common carp)
intoxicated with cypermethrin (42). These results in birds and fish
suggest the possibility of applying the present electrometric method
for biomonitoring of environmental contamination with
anticholinesterase compounds by assessing plasma or tissue
cholinesterase inhibition in wild birds which are frequently exposed
to insecticides as a result of human activities (1,16,47).
Furthermore, cholinesterase measurement in domestic animals has been
advocated as a potential biomonitoring tool for organophosphate
exposure (14,48,49).
OTHER
APPLICATIONS
Another important
application of the present cholinesterase method could be evaluation
of the enzyme activity in the amniotic fluids. This would be highly
economic screening test as cholinesterase determination in amniotic
fluids can provide an index of suspected fetal neural tube defects.
CONCLUSION
The described
electrometric method could be an added simple and practical method
for measuring blood or tissue cholinesterase activity in man and
animals exposed to organophosphate and carbamate insecticides. The
method has the potential for application in biomonitoring of
environmental exposure of wild birds or domestic animals to
anticholinesterase pesticides.
ACKNOWLEDGEMENTS
Presented in part
at the International Environmental Health Conference, Health and the
Environment in Iraq: Status, Needs and Challenges, September 19-22,
2005, Amman, Jordan.
Research works
from our laboratory were supported by the College of Veterinary
Medicine, University of Mosul.
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Table 1
Cholinesterase activity
determined by the described electrometric method in man and animals
Species |
Sex |
Cholinesterase |
Incubation time (minutes) |
Activity (∆pH) |
Reference |
Man
|
male
female |
plasma
erythrocyte
whole blood
plasma
erythrocyte
whole blood
|
20
20
20
20
20
20 |
1.05
1.18
1.25
0.91
1.19
1.23 |
27,37 |
Mice |
female |
whole blood
brain
liver
|
30
30
30 |
1.10
0.39
0.39 |
26 |
Rats |
male
female
female |
plasma
erythrocytes
brain
plasma
erythrocytes
plasma
erythrocytes
brain
|
30
30
30
30
30
30
30
30 |
0.29
0.29
0.19
0.26
0.30
0.40
0.28
0.26 |
29
28
30
|
Sheep |
mixed
male
female
|
plasma
erythrocytes
plasma
erythrocytes
plasma
erythrocyte
|
30
30
30
30
30
30 |
0.09
0.70
0.21
0.63
0.19
0.62 |
21
39 |
Goats |
mixed
male
female
male
female |
plasma
erythrocytes
plasma
erythrocytes
plasma
erythrocytes
plasma
erythrocytes
plasma
erythrocytes
|
40
40
40
40
40
40
40
40
40
40 |
0.19
0.42
0.22
0.43
0.19
0.36
0.22
0.54
0.22
0.44 |
31,32
39 |
Cattle |
male
female
|
plasma
erythrocytes
plasma
erythrocytes
|
20
20
20
20 |
0.10
0.91
0.19
0.86 |
39 |
Chickens |
mixed-chicks
male
mixed |
plasma
brain
liver
muscle
plasma
plasma
brain
liver
|
30
30
30
30
30
30
30
30 |
0.56
0.34
0.15
0.12
0.54
0.47
0.25
0.21 |
34
33
35 |
Rock dove |
mixed |
plasma
brain
liver
muscle
|
30
30
30
30 |
1.28
0.59
0.12
0.08 |
36 |
Pin tailed sandgrouse |
Mixed |
plasma
brain
liver
muscle
|
30
30
30
30 |
1.81
0.37
0.06
0.07 |
36 |
Quail |
Mixed |
plasma
brain
liver
muscle
|
30
30
30
30 |
1.23
0.39
0.19
0.06 |
36 |
Starling |
Mixed |
plasma
brain
liver
muscle
|
30
30
30
30 |
1.10
0.24
0.08
0.08 |
36 |
Fish (Cyprinus carpio) |
Mixed |
brain |
30 |
0.60 |
42 |
The cholinesterase values are mean activities
reported in the cited literature. The SE or SD of the mean
cholinesterase values can be found in the references cited.
Figure 1: Steps for the
electrometric determination of blood cholinesterase activity |
3 ml distilled water
+
0.2 ml aliquot of plasma,
erythrocytes or whole blood
+
3 ml barbital-phosphate buffer
(pH 8.1)
Measure pH (pH1)
Add 0.1 ml 7.1% acetylcholine
iodide
Incubate at 37 ºC (e.g. 20 min
in man, 30 min in birds)
Measure pH (pH2)
Cholinesterase
activity (∆
pH/incubation time)=
pH1-pH2- (∆ pH of blank, without sample)
|
Figure 2: Steps for the electrometric
determination of tissue cholinesterase activity |
Homogenize tissue (e.g. brain,
liver)
100 mg wet weight/3 ml
barbital-phosphate buffer (pH 8.1)
3 ml distilled water
+
0.2 ml aliquot of tissue
homogenate
+
3 ml barbital-phosphate buffer
(pH 8.1)
Measure
pH (pH1)
Add 0.1 ml 7.1% acetylcholine
iodide
Incubate at 37 ºC for 30 min
Measure pH (pH2)
Cholinesterase activity (∆
pH/30 min) = pH1-pH2- (∆
pH of blank, without tissue
sample)
|
|
|