Determination of serum cholinesterase activity by liquid chromatography with electrochemical detection.
A sensitive enzymatic assay to measure cholinesterase activity in serum using liquid chromatography with electrochemical detection has been devised and used to examine cholinesterase inhibition in mice treated with diisopropyl phosphorofluoridate. Acetylcholine was used as substrate, and a postcolumn reactor containing immobilized choline oxidase converted the enzymatic product, choline, and the internal standard, ethylhomocholine, into the electrochemically active H2O2. The postcolumn reactor also contained acetylcholinesterase to allow the indirect detection of the substrate. Assay optimization included investigations of substrate concentration, buffer pH and ionic strength, enzyme concentration, incubation time, and reaction termination method. The optimized procedure is applicable to samples with activities of 0.11 to 269 mmol/ml/h. Intrasample coefficient of variation for mouse serum samples was 1.7% (n = 12), while intersample coefficient of variation was 8.0% (n = 5). The mean +/- SE serum cholinesterase activity found for controls and mice treated with diisopropyl phosphofluoridate (6.3 mg/kg, ip, 24 h prior) was 158.7 +/- 5.7 mumol/ml/h and 36.6 +/- 3.1 mumol/ml/h, respectively (P less than 0.001).
Miller
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Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019.
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D000110:Acetylcholinesterase / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000818:Animals; D002021:Buffers; D002794:Choline / Q000031:analogs & derivatives / Q000378:metabolism; D002802:Cholinesterases / Q000097:blood*; D002853:Chromatography, Liquid / Q000379:methods; D004563:Electrochemistry / Q000379:methods; D004800:Enzymes, Immobilized / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007531:Isoflurophate / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D051379:Mice; D009994:Osmolar Concentration; D015203:Reproducibility of Results; D013997:Time Factors
D002021:Buffers; D004800:Enzymes, Immobilized; C042741:N-ethylhomocholine; D007531:Isoflurophate; D006861:Hydrogen Peroxide; D000429:Alcohol Oxidoreductases; C019888:choline oxidase; D000110:Acetylcholinesterase; D002802:Cholinesterases; D002794:Choline