ESTIMATION OF CHOLINE ESTERS IN BRAIN BY A NEW GAS CHROMATOGRAPHIC PROCEDURE

A new gas chromatographic procedure for microestimation of choline esters has been successfully applied to extracts of rat brain. Brains are rapidly removed after decapitation and homogenized in 85% acetone, 15% 1 N formic acid containing hexyltrimethylammonium bromide as an internal standard. After centrifugation and extraction with ether, quaternary ammonium compounds are precipitated with ammonium reineckate and the chlorides are recovered in anhydrous methanol by treatment with Biorex 9 ion-exchange resin. The residue after evaporation is allowed to react with sodium benzenethiolate in anhydrous butanone. The resulting tertiary amines are analyzed by gas chromatography as previously described,‡. Acetylcholine levels determined in this way agree with previous reports in the literature and with concurrent bioassays on frog rectus abdominis. Acetylcholine-like activity of rat brain assayed on this preparation can therefore be quantitatively accounted for by its acetylcholine content, and the results provide no support for the view that other pharmacologically active agents contribute significantly to it. Propionylcholine and butyrylcholine may be simultaneously determined by this procedure and could not be detected in fresh rat brain extracts, although both esters yielded the expected Chromatographic peaks when added to the homogenate in a total quantity of 2 nmoles. © 1969.