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Inhibitory effects of the class III antiarrhythmic compound D/L-sotalol on acetylcholinesterase (AChE; EC 3.1.1.7) isoenzymes of both erythrocytes and the human caudate nucleus and on serum cholinesterase (ChE; EC 3.1.1.8) were studied in vitro using a spectrophotometric kinetic assay with acetylthiocholine (ASCh) as substrate. Sotalol concentrations in the assays varied from 0.32 to 3.2 mM. All isoenzymes studied were inhibited by D/L-sotalol in a reversible and concentration-dependent manner. Double reciprocal plots of the reaction velocity against varying ASCh concentrations revealed that D/L-sotalol reduced substrate affinity (apparent Michaelis constant, KM, increased) of serum ChE, but did not change the enzyme's maximal rate of ASCh hydrolysis (Vmax). Thus, D/L-sotalol inhibition of serum ChE was of the competitive type (rate constant for reversible competitive inhibition: Ki = 0.51 mM). In contrast, D/L sotalol reduced the maximal reaction velocity of the AChE isoenzyme from the central nervous system (caudate nucleus), but had no influence on substrate affinity of the enzyme (KM with ASCh unchanged) indicating purely non-competitive inhibition kinetics (rate constant of reversible non-competitive inhibition: Ki = 0.44 mM). D/L-sotalol inhibition of erythrocyte AChE was of mixed competitive/non-competitive type (Ki = 0.31 mM, Ki = 0.49 mM). Non-competitive D/L-sotalol inhibition of caudate nucleus AChE and the non-competitive component of erythrocyte AChE inhibition cannot be overcome by increased concentrations of the cholinergic transmitter acetylcholine (ACh). Peak D/L-sotalol plasma levels as described in the literature for both humans (15 microM) and experimental animals (dogs: 18 microM; rats: 260 microM) as well as maximal myocardial concentrations of the substance (dogs: 46 microM; rats: 478 microM) are in the range of about 2% to 100% of the sotalol inhibition rate constants determined in the present paper for cholinesterase isoenzymes in vitro. Thus, D/L-sotalol inhibition of ACh hydrolysis in vivo may contribute to both the well known antiarrhythmic potential and proarrhythmic side effects of the compound.

The kinetics of time- and concentration-dependent covalent organophosphorus inhibition of carboxylesterase isoenzymes (EC 3.1.1.1) and cholinesterase isoenzymes (EC 3.1.1.7 and EC 3.1.1.8) were investigated using a wide range of organophosphate inhibitor concentrations (10(-10)-10(-3) mol/l) and different inhibition times. Computerized analysis of inhibition curves by weighted non-linear least-squares curve fitting was compared to graphic analysis by iterative elimination of exponential functions. Possible experimental errors due to inhibitor saturation kinetics and enzymatic organophosphate hydrolysis were thoroughly investigated. In mammalian heart muscle, three different cholinesterase isoenzymes were identified. High sensitivity and specificity of the classic differential inhibition test for carboxylesterase activity of hen brain neuropathy target esterase (NTE) could be confirmed independently with both methods of inhibition curve analysis.

Carboxylesterases (EC 3.1.1.1) of chicken brain were investigated by applying kinetic analysis of organophosphorus inhibition. By iterative elimination of exponential inhibition curves and by sequential inhibition experiments using a combination of two organophosphorus inhibitors, 11 different carboxylesterases of chicken brain were characterized with respect to their phenyl valerate-hydrolyzing activity (milliunits per gram of brain) and their inhibition by O,O-diethyl O-4-nitrophenyl phosphate (Paraoxon), O,O-diisopropylphosphorofluoridate, and N,N'-diisopropylphosphorodiamidic fluoride (Mipafox). The bimolecular inhibition rate constants (liters . mole-1 . min-1) were calculated for the 11 enzymes and 3 organophosphorus compounds. The corresponding data for acetylcholinesterase (EC 3.1.1.7) in chicken brain were determined. The importance of inhibition rate constants for the development of acute cholinergic symptoms, delayed neurotoxicity, and atypical organophosphate effects is shown.

Carboxylesterase activity of primate brain (Macaca mulatta) was determined using phenyl valerate (PV) as substrate. Eight carboxylesterases of primate brain were characterized in respect to PV-hydrolysing activity and to their inhibition rate constants for the reaction with organophosphorus compounds. Carboxylesterase III was identified as neurotoxic esterase (NTE). Organophosphate inhibition data of primate acetylcholinesterase (EC 3.1.1.7) and of primate cholinesterase (EC 3.1.1.8) were determined and are compared to corresponding data of primate brain carboxylesterases. Physiological functions, clinical and toxicological significance of primate brain carboxylesterases are discussed.

A standard procedure for lipid-extraction of lyophilized hen brain material is described. Nine carboxylesterase isoenzymes (EC 3.1.1.1) are identified in lipid-extracted lyophilized material (LELM) using kinetic analysis of organophosphate inhibition. Total phenyl valerate (PV) hydrolysing carboxylesterase activity in LELM is 43.3 U X g-1. Two carboxylesterase isoenzymes of LELM are classified as neurotoxic esterases (NTEA and NTEB). Using n-octylglucoside 51% of the water-insoluble neurotoxic esterase activity from LELM are solubilized. Six carboxylesterase isoenzymes including NTEA (6.5 U X 1(-1] and NTEB (4.2 U X 1(-1] are present in the solubilized preparation. Throughout purification and separation steps carboxylesterase isoenzymes are identified by their rate constants for the reaction with organophosphorus inhibitors.

Two phenyl valerate hydrolyzing carboxylesterases (EC 3.1.1.1) of hen brain were identified as neurotoxic esterases (NTEA and NTEB) by kinetic analysis of organophosphorus inhibition curves. The activities of both NTE isoenzymes with phenyl valerate (PV) as substrate and their inhibition rate constants were determined in six different animals. In-vivo-application of a single oral dose of 500 mg/kg triorthocresyl phosphate (TOCP) caused 86% inhibition of NTEA and 93% inhibition of NTEB within 24 h. Total NTE activity (NTEA plus NTEB) determined by kinetic analysis shows an excellent correlation (r = 0.989) to NTE activity simultaneously tested with a differential NTE assay. The excellent sensitivity (97%) and high specificity (79%) of the NTE differential test is demonstrated.

Cholinesterases in hen brain were characterized with respect to inhibition kinetics and substrate specificity. Three organophosphorus inhibitors were used: diethyl p-nitrophenyl phosphate (Paraoxon, E 600), di-isopropylphosphorofluoridate (DFP), and N,N'-di-isopropylphosphorodiamidic fluoride (Mipafox). The kinetics of irreversible cholinesterase inhibition were studied using two substrates, acetylthiocholine and butyrylthiocholine. The inhibition curves were analysed by the method of iterative elimination of exponential functions. Final classification of the different enzymes was done by combining two inhibitors in sequential inhibition expts. Six cholinesterases were shown to hydrolyse choline esters in hen brain, one was identified as acetylcholinesterase (EC 3.1.1.7) and one as cholinesterase (EC 3.1.1.8). Four enzymes can be classified as intermediate type cholinesterases according to their substrate specificity and to their inhibition constants. The possible role of different brain cholinesterases for the development of atypical symptoms following organophosphate intoxication is discussed.

Carboxylesterase activity (EC 3.1.1.1) of hen brain including neurotoxic esterases NTEA and NTEB is solubilized from lyophilized lipid-extracted brain material by the use of n-octylglucoside. The solubilized enzymes are subjected to free isoelectric focusing, six carboxyl - esterase activity peaks are obtained. By gel filtration on Sephacryl S-300 neurotoxic esterases are separated from carboxylesterase isoenzymes V and X. The molecular weight of the neurotoxic esterases is estimated to be 1.8 X 10(6).