Frequent attenders in general practice: a study from Slovenia

This study describes the entrapment of squid-type diisopropylphosphorofluoridate-hydrolyzing enzyme (DFPase) within mouse red blood cells. These erythrocytes thereby gain the ability to rapidly hydrolyze alkylphosphate cholinesterase (ChE) inhibitors such as diisopropyl fluorophosphate (DFP). DFPase rapidly hydrolyzes DFP to diisopropyl phosphate. Resealed erythrocytes provide a stable carrier system that can preserve the activity of encapsulated enzymes against otherwise rapid in vivo degradation; thus, ChE inhibitors can be degraded to relatively nontoxic metabolites by these erythrocyte carriers. Squid DFPase was purified from the hepatopancreas of Atlantic squid and DFPase activity was determined by measuring changes in fluoride ion concentration using a fluoride ion selective electrode. Mouse erythrocytes in suspension with excess squid DFPase were dialyzed against hypotonic buffer to allow the encapsulation of the enzyme to occur. Cells were then resealed by returning the suspension to isosmotic with saline. Rate of DFP hydrolysis observed with these cells was much greater than the rate of nonenzymatic hydrolysis and was directly proportional to the amount of the erythrocyte suspension added to the assay solution. The rate of hydrolysis was first order in substrate. Erythrocyte controls showed no endogenous DFPase activity. These results suggest that enzyme entrapment may be developed as a method to prevent and antagonize organophosphate poisoning.