Evaluation of probable interaction of Pralidoxime in cholinesterase activity assay by colorimetric method in human peripheral blood
Trauma Monthly: 13 (4); 287-292 Article Type: Research Article
M. Evaluation of probable interaction of Pralidoxime in cholinesterase activity assay by colorimetric method in human peripheral blood,
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AIMS: Evaluation of probable interaction between Pralidoxime and thiocholine substrates by colorimetric Ellman's method and its effect on cholinesterase activity assay in human peripheral blood.
METHODS: 10 human peripheral blood samples were collected. We compared effect of Pralidoxime (PRX) and enzymes (ENZ) on Acetyl-thiocholine (ATC) and Butyryl-thiocholine (BTC) by Ellman's method at 37°C. For each enzyme, absorbance of two experiment groups (Enzyme group [Enz(50µl+Sub(2mM)] and PRX group [PRX(100µM)+Sub(2mM)]) were read at 410nm against suitable control (all substances in test tube except enzyme and Pralidoxime) and used in enzyme activity calculation. Data analyzed by Kruskal-Wallis test by Prism 5 software. We repeated PRX group with 3, 10, 30, 100 and 300 µM of pralidoxime in 1 up to 5 minute and calculated relation between false activity (without any enzyme) and PRX concentration or reaction times by correlation analysis.
RESULTS:False activity due to PRX and ATC interaction in Ellman’s method was 19.4±3.736% of AChE activity. False activity due to PRX and BTC interaction in Ellman’s method was 8.2±1.474% of BChE activity. False activity had direct correlation with PRX concentration (r>0.89) and reverse correlation with reaction time (r<-0.9).
CONCLUSION: Pralidoxime ability to dose and time dependent hydrolysis of thiocholine substrates, seems to increases cholinesterase activity falsely. So Ellman’s method test has some limitation for cholinesterase activity assay in presence of Pralidoxime and usage of this test for concentrations more than 200µM of Pralidoxime is not recommended.
Human Peripheral Blood; Acetylcholinesterase; Butyrylcholinesterase; Pralidoxime; Ellmans Method; False Activity
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