A NOVEL METHOD THAT MARKEDLY INCREASES THE SENSITIVITY OF THE ERYTHROCYTE ACETYLCHOLINESTERASE ASSAY, SUITABLE FOR USE IN PESTICIDE-TREATEDRATS

Although the specific activity of rat erythrocyte acetylcholinesterase activity is reasonably high, use of the standard spectrophotometric a ssay presents special problems due primarily to the interference of he moglobin with the absorbance spectrum of the assay product, 5-thio-2-n itrobenzoic acid. To limit the hemoglobin interference, the erythrocyt e sample is diluted at least 20- to 25-fold before assay, but this dil ution decreases the level of measured activity. We have sought to incr ease the sensitivity of the rat erythrocyte acetylcholinesterase assay by employing a standard technique to release [using phosphatidylinosi tol-specific phospholipase C (PIPLC)] the acetylcholinesterase molecul es from the erythrocyte surface without lysis of the erythrocytes. The present group of studies determined if the inhibition of the acetylch olinesterase activity that had been stripped off the erythrocytes take n from pesticide-treated rats reflected the acetylcholinesterase inhib ition in the unstripped erythrocytes of the same animals. In rats trea ted with graded dosages of an organophosphate (fenthion, paraoxon, or chlorpyrifos) or a carbamate (carbaryl), the acetylcholinesterase inhi bition in the released fraction mimicked the inhibition in the convent ional erythrocyte sample. In control animals, use of this released fra ction increased the sensitivity of the erythrocyte acetylcholinesteras e assay by at least 10-fold. In conclusion, utilizing PIPLC to release (i.e., strip) and separate the erythrocyte acetylcholinesterase activ ity from the interfering hemoglobin may be a convenient method for mar kedly raising the sensitivity of the rat erythrocyte acetylcholinester ase assay and predicts erythrocyte acetylcholinesterase inhibition in animals treated with cholinesterase-inhibiting pesticides.