© 1991 Oxford University Press
research-article |
A Microassay Method for Neurotoxic Esterase Determinations1
Virginia-Maryland Regional College of Veterinary Medicine Blacksburg, Virginia 24061
Received March 12, 1990; accepted August 13, 1990
A microtiter plate reader with an associated computer to average triplicate samples and subtract blanks was used for reading and calculating neurotoxic esterase (NTE, also known as neuropathy target esterase) activities in spinal cord regions of hens 4 hr after administration of diisopropylphosphorofluoridate (DFP, 0.5 mg/kg sc). Although NTE inhibition is an early indicator of organophosphorus ester-induced delayed neuropathy, DFP-induced inhibition was not greater in regions of the spinal cord where pathological changes are most notable. Acetylcholinesterase (AChE) activities and protein determinations were also done on these tissues using microassay methods. DFP-induced AChE inhibition was similar to NTE inhibition. In addition to the capability to be used for small regional esterase activity measurements, the microassay was advantageous because the number of samples incorporated into a single assay was increased and the time needed for the NTE assay was reduced by 50%. Total volume of incubate in each well was 0.3 ml; the incubate contained 1/20 quantities of sample and reagents necessary in more conventional assays. Validation of the microassay was performed by comparison with more conventional assays when measuring inhibition of NTE and AChE in brains of control and experimental hens of two different genetic strains (B13B13 and B21B21 white leghorns). Experimental birds were given DFP, 0.5 mg/kg sc, 24 hr before samples were collected. NTE activities in brains of control hens were similar using both types of NTE analytical procedures. Percentage inhibition of NTE caused by DFP was within 4% using both assay procedures in both strains of hens. The microassay was sensitive enough to detect NTE activity in 42 µg of hen brain after 15 min of incubation. Hen lymphocytes could also be examined for effects of organophosphorus esters on NTE activity, with 14.1 ±2.2 and 8.3 ±2.2 /xmol/15 min/mg protein in 1x106 cells measured in samples taken before and 4 hr after administration of 0.5 mg/kg SC DFP.