Development of a dynamic model for real-time determination of membrane-bound acetylcholinesterase activity upon perfusion with inhibitors and reactivators.
Quantitative predictions of the course of acetylcholinesterase (AChE) activity, following interference of inhibitors and reactivators, are usually obscured by the time-dependent changes of all reaction partners. To mimic these dynamics we developed an in vitro model. Immobilized human erythrocyte ghosts in a bioreactor were continuously perfused while AChE activity was monitored by a modified Ellman method. The perfusion system consisted of two HPLC pumps with integrated quaternary low-pressure gradient formers that were programmed by a computer using commercial HPLC software. The combined eluates passed a particle filter (Millex-GS, 0.22 microm) containing a thin layer of erythrocytes that was immersed in a temperature-controlled water bath. The effluent passed a flow cell in a UV-vis detector, the signal of which was digitized, written to disc and calculated with curve fitting programs. AChE activity decreased by 3.4% within 2.5 h. The day-to-day variation of the freshly prepared bioreactor using the same enzyme source was +/-3.3%. Residual activity of 0.2% marked the limit of quantification. Following perfusion with paraoxon, pseudo first-order rate constants of inhibition were established that did not differ from results obtained in conventional assays. The same holds true for reactivation with obidoxime. The set-up presented allows freely programmable time-dependent changes of up to eight solvents to mimic pharmacokinetic profiles without accumulation of products. Due to some hysteresis in the system, reaction half-lives should be >3 min and concentration changes in critical compounds should exceed half-lives of 5 min. Otherwise, the system offers much flexibility and operates with high precision.
Eckert
Saskia
S
Walther-Straub-Institute of Pharmacology and Toxicology, Ludwig-Maximilians-University of Munich, Goethestr. 33, D-80336 Munich, Germany.
Eyer
Peter
P
Mückter
Harald
H
Worek
Franz
F
D000110:Acetylcholinesterase / Q000378:metabolism*; D002800:Cholinesterase Inhibitors / Q000494:pharmacology*; D002801:Cholinesterase Reactivators / Q000494:pharmacology*; D002863:Chromogenic Compounds / Q000032:analysis; D003199:Computer Systems; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004795:Enzyme Stability; D004867:Equipment Design; D004910:Erythrocyte Membrane / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D009768:Obidoxime Chloride / Q000494:pharmacology; D010261:Paraoxon / Q000494:pharmacology; D010477:Perfusion / Q000295:instrumentation / Q000379:methods; D015203:Reproducibility of Results; D013053:Spectrophotometry / Q000295:instrumentation / Q000379:methods; D013696:Temperature; D013997:Time Factors
D002800:Cholinesterase Inhibitors; D002801:Cholinesterase Reactivators; D002863:Chromogenic Compounds; D009768:Obidoxime Chloride; D004228:Dithionitrobenzoic Acid; D000110:Acetylcholinesterase; D010261:Paraoxon