2.4.1. Protocol for Measurement of Acetylcholinesterase (AChE) Activity

The activity of AChE in the plasma and S9 samples was determined according to the method of Ellman et al. [38]. For the plasma samples, the reaction mixture contained 5 µL plasma diluted 5x with phosphate buffer (0.1 M, pH 7.2), 180 µL phosphate buffer (0.1 M, pH 7.2), 10 µL DTNB (1.6 mM, prepared with phosphate buffer (0.1 M, pH 7.2)), and 10 µL acetylthiocholine iodide (156 mM, prepared with distilled water). Increase in absorbance was measured for 5 min at 412 nm. For the S9 samples, the reaction mixture contained 25 µL S9 diluted 10x with phosphate buffer (0.1 M, pH 7.2), 180 µL phosphate buffer (0.1 M, pH 7.2), 10 µL DTNB (1.6 mM prepared with phosphate buffer (0.1 M, pH 7.2)), and 10 µL acetylthiocholine iodide (156 mM, prepared with distilled water). Increase in absorbance was measured for 10 min at 412 nm. Blank measurements of the plasma and S9 were performed in parallel containing 180 µL phosphate buffer, 10 µL DTNB, and 10 µL acetylthiocholine iodide (all prepared in the same way as described previously). Specific enzyme activity was calculated with the extinction coefficient (ε) = 13.6 × 10³ M⁻¹ cm⁻¹.