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Validation of an Automated Spectrophotometric Assay for the Determination of Cholinesterase Activity in Canine Serum

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The determination of enzymatic activity of cholinesterase is a useful diagnostic method to detect exposure to anticholinesterase compounds in human and in veterinary medicine. We validated a modification of the Ellman method in canine serum and applied it to the diagnosis of dogs poisoned with anticholinesterase substances. The method used butyrylthiocholine as substrate and potassium hexacyanoferrate as chromophore. The reference range calculated on 60 clinically healthy dogs was set between 3405 and 6561 U/L (chi-square test for normal distribution, p > 0.05). The overall mean intra-assay and inter-assay coefficients of variation were 0.53% and 3.83%, respectively. The assay was linear when using two sera with 12 538 U/L and 6604 U/L serum cholinesterase activity (r2=0.997 and 0.999, respectively). The mean recovery values of pooled sera with a mean pseudocholinesterase (PChE) activity of 12 081 U/L and pooled sera with a mean PChE activity of 3415 U/L were 103.5% and 102.8%, respectively. Six dogs with a diagnosis of anticholinesterase compound intoxication showed a decrease in cholinesterase activity of at least 50% of normal activity with a mean ± SD of 487 ± 291 U/L ranging from 169 to 847 U/L. This technique conforms to the current standard for precision, linearity and accuracy and is a useful method for the complementary diagnosis of organophosphate or carbamate insecticide intoxication in dogs.

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coefficient of variation


5,5’-dithiobis(nitrobenzoic acid)


standard deviation




pseudocholinester- ase

r :

correlation coefficient (Pearson)

r2 :

square of correlation coefficient


serum protein electrophoresis


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Correspondence to L. Solano-Gallego.

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Furlanello, T., Simonato, G., Caldin, M. et al. Validation of an Automated Spectrophotometric Assay for the Determination of Cholinesterase Activity in Canine Serum. Vet Res Commun 30, 723–733 (2006). https://doi.org/10.1007/s11259-006-3354-9

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  • canine serum
  • cholinesterase
  • hexacyanoferrate
  • organophosphate compounds
  • spectophotometric assay