Abstract
This investigation effort is focused on the new conceptual approach to antagonize organophosphorus (OP) intoxication. This was accomplished by the use of resealed carrier erythrocytes (CRBC) containing a recombinant enzyme derived from Flavobacterium and expressed in Escherichia coli. The mechanism of the CRBC apparently was to degrade the OPs absorbed into the blood before it can exert its systemic toxic effect. Since there were no methods reported to determine organophosphorus acid (OPA) anhydrase activity in blood, it was necessary to develop a method as hemoglobin interfered with all spectrophotometric determinations. This was accomplished by extracting the reaction products, 4-nitrophenol with dichloromethane and determining 4-nitrophenol after extraction colorimetrically. Subsequently a more convenient continuous method without solvent extraction was developed which permitted conducting kinetic studies on this enzyme. Enzyme from cultured E. coli cells was isolated and purified over 1600 folds. The purified OPA anhydrase was successfully encapsulated within carrier erythrocytes by hypotonic dialysis. Enzymatic characteristics of the encapsulated OPA anhydrase were investigated. As diffusing through the cell membrane, paraoxon was readily degraded by the entrapped enzyme within CRBC. This enzyme apparently followed Michaelis-Menton kinetics. When these CRBCs were incubated with paraoxon, diethylphosphate and 4-nitrophenol formed rapidly indicating these cells could rapidly degrade paraoxon. These resealed erythrocytes then were administered to mice and they were found to be extremely effective in antagonizing the lethal effect of paraoxon. These CRBCs were much more effective than the classic antidotal combination of atropine and pralidoxime (2-PAM). Moreover, these cells actually destroy the OP, whereas atropine and 2-PAM do not. The CRBCs provided a striking massive synergistic protective effect to the lethal effect of paraoxon. Animals receiving the combinational treatment of CRBC, atropine and 2-PAM can be protected against well over 1,000 LD50 doses of paraoxon. This combination of CRBC, atropine and pralidoxime is far more efficacious than any OP antidotal system ever reported.
Pei, Luqi (1994). Antagonism of organophosphorus intoxications with resealed erythrocytes containing organophosphorus acid anhydrase. Texas A&M University. Texas A&M University. Libraries. Available electronically from
https : / /hdl .handle .net /1969 .1 /DISSERTATIONS -1551985.