The determination of serum cholinesterase activity was based on the hydrolysis of butyrylcholine chloride, solvent extraction, and the quantitative estimation of the hydrolytic product, butyric acid, by gas-solid chromatography. Using this procedure, optimum conditions of pH and buffer strength for the cholinesterase activity were pH 8.0 and 0.05 M Tris-HC1, respectively. The cholinesterase activities in blood samples from healthy subjects, as determined by this method, were in the range of normal values reported in the literature. The effects of eserine and other drugs on the cholinesterase activity were also reported. The high accuracy (1.1%) and high sensitivity which permit the determination of butyric acid at levels down to 1 pg/ml make this procedure attractive as a serum cholinesterase assay.
In most analytical procedures for assaying serum cholinesterase activity, the extent of enzymatic activity is determined either titrimetrically (1,2), mannometrically
(3), photometrically (4-Q, or fluorometrically (9). A recently reported te.chnique for the determination of estereolytic activity, based on enzymatic hydrolysis of an ester, solvent extraction, and direct quantitative estimation of the hydrolytic product, fatty acid, by gas-solid chromatography (GSC), marked an important advancement in the refinement of methods (10). The advantages of this technique are high sensitivity and high reproducibility.
However, the gas chromatographic procedure reported needs some modifications since it lacks the specificity required for accurate asay of serum cholinesterase. The main objective of this paper was to devise a modified gas chromatographic technique suitable for accurate determination of cholinesterase activity in blood serum.
Methods
Materials. Butyrylcholine chloride of analytical grade, purchased from Koch-Light Laboratories, was further purified as follows: Traces of butyric acid were removed by treatment of a 1 M aqueous solution of butyrylcholine chloride (10 ml) with 1 M acetic acid (1 ml). The mixture was extracted three times with diethyl ether (30 ml). The residual ether in the aqueous solution was evaporated off and diluted with buffer to a final