A novel method that markedly increases the sensitivity of the erythrocyte acetylcholinesterase assay, suitable for use in pesticide-treated rats

Although the specific activity of rat erythrocyte acetylcholinesterase activity is reasonably high, use of the standard spectrophotometric assay presents special problems due primarily to the interference of hemoglobin with the absorbance spectrum of the assay product, 5-thio-2-nitrobenzoic acid. To limit the hemoglobin interference, the erythrocyte sample is diluted at least 20- to 25-fold before assay, but this dilution decreases the level of measured activity. We have sought to increase the sensitivity of the rat erythrocyte acetylcholinesterase assay by employing a standard technique to release [using phosphatidylinositol-specific phospholipase C (PiPLC)] the acetylcholinesterase molecules from the erythrocyte surface without lysis of the erythrocytes. The present group of studies determined if the inhibition of the acetylcholinesterase activity that had been stripped off the erythrocytes taken from pesticide-treated rats reflected the acetylcholinesterase inhibition in the unstripped erythrocytes of the same animals. In rats treated with graded dosages of an organophosphate (fenthion, paraoxon, or chlorpyrifos) or a carbamate (carbaryl), the acetylcholinesterase inhibition in the released fraction mimicked the inhibition in the conventional erythrocyte sample. In control animals, use of this released fraction increased the sensitivity of the erythrocyte acetylcholinesterase assay by at least 10-fold. In conclusion, utilizing PiPLC to release (i.e., strip) and separate the erythrocyte acetylcholinesterase activity from the interfering hemoglobin may be a convenient method for markedly raising the sensitivity of the rat erythrocyte acetylcholinesterase assay and predicts erythrocyte acetylcholinesterase inhibition in animals treated with cholinesterase-inhibiting pesticides. © 1995 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.

Duke Scholars

Author Stephanie Padilla Environmental Sciences and Policy