Monitoring blood (whole blood, plasma, serum or erythrocytes) cholinesterase (ChE) activity is used to assess the exposure of man and animals to organophosphate and carbamate insecticides (1-3). Usually a reduction of blood ChE activity by 20-30% from baseline values is an indication of exposure to ChE inhibiting pesticides (2,3). Enzyme inhibition by > 50% calls for immediate healthcare measures (1-3). Several methods are available to measure ChE activity in the blood and nervous tissues (3,4). A modified electrometric method is available for measuring blood and brain ChE activity that is a simple procedure which does not need elaborate equipment and was adopted by many researchers (5-27). However, a published article erroneously referenced this modified electrometric method mentioned above (28). This prompted us to clarify and elucidate few key points about the method. The original electrometric Michel method has two incubation periods for a total of more than one hour at 25 oC (29), whereas our electrometric method is mainly characterized by a single incubation period of 20 min in man (5) and 30 min or even more in different animal species at 37 oC (6-27).
Our electrometric method of ChE determination is a modified one that we refined and developed after several years of research and validations in various animal species (6-27) as well as in man (5,25). We also applied the developed method on many research projects on poisoning with organophosphate and carbamate insecticides (6-26). For review of differences in animal species and man, see published references (5,18,23-26,30).
Because of the importance of the subject of ChE biomonitoring (1-4) and to further clarify the assay technique of the modified electrometric method, here is a brief but concise description of the procedure. It is hoped that it would be beneficial for researchers of limited resources.
Chemicals: 7.5% aqueous solution of acetylthiocholine iodide (or 7.1% acetylcholine iodide), sodium barbital, potassium dihydrogen phosphate, sodium chloride, and double distilled water.
Barbital-Phosphate buffer: 1.237 g sodium barbital, 0.163 g potassium dihydrogen phosphate and 35.07 g sodium chloride/ L distilled water, pH 8.1.
Equipment: Centrifuge, Water bath, pH meter
- Collect venous blood samples in heparinized test tubes.
- Separate the plasma from erythrocytes by centrifugation.
- To a 10-ml beaker add 3 ml distilled water, 0.2 ml plasma, erythrocytes or even whole blood.
- Add 3 ml of barbital-phosphate buffer solution.
- Measure the pH of the mixture (pH1).
- Add 0.10 ml of 7.5% acetylthiocholine iodide (or 7.1% acetylcholine iodide) to the mixture.
- Incubated at 37 ºC for 30 min for most animal species; 20 min in man.
- Measure the pH of the mixture (pH2).
- Calculate cholinesterase activity:
ChE activity (DpH/30 min or 20 min) = (pH1 – pH2) - D pH of blank
The blank is without sample. The unit of activity is Δ pH/30 or 20 min.