COMPARISON OF ASSAY METHODS FOR STUDYING O,O-DIETHYL O-P-NITROPHENYL PHOSPHATE (PARAOXON) DETOXICATION IN VITRO

Three different techniques were used to study the inactivation of paraoxon (O, O-diethyl, O-p-nitrophenyl phosphate) by animal tissues in vitro. The first procedure (spectrophotometric technique) measures the amount of p-nitrophenol released during the hydrolysis of paraoxon (4 × 10-4 M). The second technique estimates the hydrolysis of paraoxon (7·7 × 10-3 M) by manometric measurement of CO2 liberated in a bicarbonate buffer (manometric technique). The third procedure (anti-CHE technique) measures the loss of anticholinesterase (anti-CHE) activity of paraoxon (1 × 10-7 M) after incubation with tissues. The kinetics of the reactions, tissue distributions, sex and species comparisons, the effects of calcium, magnesium and EDTA, and the effects of pretreatment in vivo with organophosphate esters were studied. The influence of these factors differed depending upon whether the inactivation of paraoxon was measured by the spectrophotometric or the anti-CHE technique. This indicated that the inactivation mechanism operating in the anti-CHE system was not the same as paraoxon hydrolysis as measured by the spectrophotometric and manometric techniques. The results of studies of the effects of incubation time, substrate concentration, dialysis, cofactors and pretreatment with organophosphate esters suggest that tissue binding of paraoxon is the principal mechanism of inactivation operating in the anti-CHE system. © 1969.