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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: fouadmohammad@yahoo.com)

 


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)

 

 

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