Estimation of Cholinesterase Activity (EC 3.1.1.7; 3.1.1.8) in Undiluted Plasma and Erythrocytes as a Tool for Measuring In Vivo Effects of Reversible Inhibitors

Abstract

Summary:In vivo effects of reversible inhibitors of cholinesterase activity were determined radiometrically in undiluted samples of erythrocytes and plasma. [ 14 C]acetylcholine at substrate saturation, 25 °C and pH 7.4 permitted rapid and precise determination of butyrylcholinesterase (EC 3.1.1.8) and acetylcholinesterase (EC 3.1.1.7) activities. Reference values for acetylcholinesterase and butyrylcholinesterase were estimated in the plasma and erythrocyte haemolysate of 102 healthy volunteers.The time course of in vitro inhibition was monitored, starting immediately after addition of 9-amino-l,2,3,4-tetrahydroacridine (tacrine), eserine or pyridostigmine to undiluted human plasma. Maximal inhibition (in vitro) was seen within 60 min with tacrine and eserine, in contrast to 180min with pyridostigmine. The inhibition remained constant for more than 10 h except with eserine, from which enzyme activity showed an early recovery. Concentration response experiments were performed in undiluted human plasma and undiluted human erythrocyte haemolysate. /^-values of tacrine, eserine and pyridostigmine were estimated. In contrast to pyridostigmine and eserine, tacrine was found to have a higher affinity for butyrylcholinesterase than for acetylcholinesterase. Tacrine at 2.5 μηιοΐ/ΐ resulted in complete inhibition of butyrylcholinesterase and 70% inhibition of acetylcholinesterase activity. Dilution of these samples up to 100-fold was accompanied by almost complete recovery of acetylcholinesterase and by 50% recovery of butyrylcholinesterase. Introduction,

Inhibition of Acetylcholinesterase Activity in Human Brain Tissue and Erythrocytes by Galanthamine, Physostigmine and Tacrine

“…Since measurement of for symptomatic treatment of cognitive deficits and red-cell acetylcholinesterase Inhibition ex vivo may memory impairment in Alzheimer's disease. One pos-predict enzyme Inhibition in the brain (6,7), it seemed tulated mechanism is restoration of the cholinergic useful to perform additional in vitro experiments with deficit at synaptic sites in the brain by inhibition of erythrocytes. Postmortem acetylcholinesterase activacetylcholine metabolisipti (1), b t severalother mech^ ity has been shown to be stable (8) for at least 31 h. anisms have also been discussed (2 -5).…”

Galanthamine Hydrobromide in a Long-Term Treatment of Alzheimer's Disease

“…The reversible nature of the enzyme-inhibitor complex used to be an experimental problem, which, however, was solved by using a method that did not require ex vivo dilution of plasma and erythrocyte samples [18].…”

“…AChE and butyrylcholinesterase activities were estimated ac cording to the radiometric method of Thomsen et al [18], which allowed for undiluted measurement ex vivo, using u C-labelled ace tylcholine iodide (NEN, Dreicich, FRG) at final substrate concen trations of 9.1 mmol/1 in plasma and 3.6 mmol/1 in red blood cells (pH 7.4, temperature 25 °C). The incubation periods were 15 s (erythrocytes) and 120 s (plasma).…”

“…These findings were confirmed in vitro by our group [unpubl. data] in plasma and erythrocytes of healthy volunteers with a radiometrical method [18]. Additional support is derived from animal studies [19] in vitro, where GAL exerted an equipotent influence on cholinesterases in erythrocytes and brain tissue but was clearly less effective in the plasma [unpubl.…”

Acetylcholinesterase-inhibitors in the treatment of autistic disorders

“…Tacrine (Cognex ® , Warner-Lambert, Canada) and physostigmine (Antilirium © , Forest Pharmaceuticals Inc., MO, USA) exhibit a higher degree of AChE inhibition than galantamine (Reminyl ® , Janssen Pharmaceuticals, NJ, USA) in these regions of the brain, for example, which can also be demonstrated on erythrocyte AChE activity [22]. All these substances inhibit AChE rather than butyrylcholinesterase in human plasma and erythrocytes and therefore, also attenuate drug-and lesion-induced cognitive deficits of learning and memory in animal models [23,24].…”