Simultaneous analysis of choline and acetylcholine levels in rat brain by pyrolysis gas chromatography

Gas chromatographic analysis of the tertiary amines resulting from either chemical (1,2) or heat-catalyzed (3,4) removal of a quarternary methyl group from choline esters has provided a sensitive chemical assay for acetylcholine (ACh) in various tissues. In order to study ACh turnover using precursor labeling techniques it is also necessary to measure the level of free choline in tissue. Recent publications on the level of choline in the central nervous system (5,6) and on the role its uptake plays in the regulation of ACh synthesis in cholinergic neurons have also stimulated interest in the measurement of choline. Methods for simultaneous analysis of choline and ACh employing chemical demethylation have previously been published (7). The present paper describes the modification of a previous method (4) which is necessary for simultaneous analysis of choline and ACh by pyrolysis gas chromatography. These modifications are required because endogenously occurring amounts of choline are not reproducibly precipitated as the eneiodide salt from aqueous solutions and choline cannot be quantitatively converted to its tertiary amine analog by pyrolysis. It is therefore quantitatively isolated and converted to a choline ester prior to gas chromatographic analysis.