Electrophoretically mediated microanalysis for the evaluation of interspecies variation in cholinesterase metabolism.

This study describes an electrophoretically mediated microanalysis method, suitable for the preclinical evaluation of the hydrolysis of ester drugs by the serum of different animals and for further characterization of human-animal correlation. Dog, cat, cow, horse, sheep, rat and human serum were diluted (25%) in the appropriate buffer and replaced the enzyme solution usually used in electrophoretically mediated microanalysis methods for the study of enzyme kinetics. They were then compared in terms of the ability to hydrolyze acetylthiocholine and butyrylthiocholine (0.25 mM) by in-capillary reaction. Human serum afforded the highest conversion rates (52% butyryltiocholine and 34% acetylthiocholine) followed by horse (31 and 35%), dog (26 and 24%), cat (22 and 14%), rat (11 and 15%) and sheep (8 and 8%). Hydrolysis by bovine serum was negligible. The method is fast (under 8 min including rinsing steps), sensitive (under 25 microM substrate could be quantified) and repeatable (RSD approximately 2%), only requiring minute amounts of sample.

Determination of whole blood cholinesterase in different animal species using specific substrates.

Whole blood cholinesterase was measured using acetyl-, butyryl- and propionylthiocholine as substrates in 10 healthy adult dogs, cats, horses, pigs, goats, sheep and cows, in order to determine and characterise the cholinesterase activity in whole blood of the main domestic animals. An in vitro exposure test with two anticholinesterase compounds, the organophosphate insecticide coumaphos and the carbamate insecticide imidocarb, was also performed. In whole blood of ruminants and pigs, acetylthiocholine yielded the highest cholinesterase activity and other substrates were poorly hydrolysed; in dogs and cats, although acetylthiocholine showed the highest cholinesterase activity, butyryl- and propionylthiocholine also produced high cholinesterase values; in horses, propionylthiocholine was the substrate that yielded the highest cholinesterase activity, closely followed by butyrylthiocholine. All within- and between-run coefficients of variation observed in whole blood samples were less than 5 and 7 per cent, respectively, except when butyrylthiocholine was used as substrate in ruminant blood samples. Butyryl- and propionylthiocholine were the substrates that yielded higher inhibitions after coumaphos exposure, whereas the use of acetylthiocholine showed the highest cholinesterase inhibition after imidocarb exposure. The use of at least two substrates (acetyl and butyrylthiocholine) is recommended for whole blood cholinesterase analyses in domestic animals since it will allow monitoring of both acetyl- and butyrylcholinesterase activities, respectively, and a more accurate detection of exposure to anticholinesterase compounds. However, acetylthiocholine could be used as a unique substrate for whole blood cholinesterase determination in porcine and ruminant samples since butyrylcholinesterase activity is very low in these species. Additionally, propionylthiocholine could be used as an alternative substrate to butyrylthiocholine in horse whole blood samples.

Optimization and kinetic studies of human erythrocyte membrane-bound acetylcholinesterase.

The human erythrocyte membrane-bound acetylcholinesterase was characterized with respect to optimal assay conditions for its kinetic properties. It was found that 40.0 micrograms protein and 5.0 min incubation time were the suitable concentration of AChE protein and reaction time for the linearity of AChE activity at 25 degrees C. The Vmax for the AChE was 35% higher in the presence of 25 mM sodium phosphate buffer than 25 mM Tris-HCl buffer. The AChE activity was assayed at various strengths of sodium phosphate buffer (0.0125-0.20 M), and the optimum strength was found to be 0.05 M. The optimum substrate (ASCh) concentration was found to be 5.0 mM whereas at higher substrate concentrations, the AChE activity declined. The ASCh concentration ranges for different orders of the reactions were determined and kinetic parameters (Km, Vmax, Kcat and Ksp) were established at each order of the reaction.

The effect of storage at different temperatures on cholinesterase activity in human serum.

The effect of storage on the catalytic concentration of cholinesterase and on the reference values of cholinesterase in human serum were studied. When serum was stored at room temperature (20 degrees C), at 4 degrees C and at - 20 degrees C (one year) there was no change in catalytic activity. Even after nine times freezing and thawing (over nine weeks) the catalytic activity was unaffected. The average reference value was significantly higher for males than for females (3.11 +/- 0.57 vs 2.50 +/- 0.43 kU/l).