Ccxviii. a Colorimetric Method for the Determination of Choline and Acetyl-

WHinE the presence of small amounts of choline and acetylcholine can easily be detected by biological methods, their quantitative micro-chemical estimation presents considerable difficulty. The only method hitherto described for the micro-chemical estimation of choline appears to be that of Roman [1930], a modification of the macro-method of Stanek [1906] depending on the precipitation of choline as choline periodide. By this method quantities of choline from 0-001 to 5 mg. can be estimated, but its application is limited since creatinine, adenine and other nitrogenous bases are also precipitated. Furthermore, the method is complicated by the fact that the physical conditions of the experiment require very careful control. In the present paper a simple colorimetric method for the estimation of choline and acetylcholine is described. The principle of the method is the same as that used by Kapfhammer and Bischoff [1930] for the isolation of choline and acetylcholine from tissues. It depends on the fact that choline and acetylcholine form reineckates which are insoluble in ice-cold water and in absolute alcohol but are easily soluble in acetone giving a bright red solution. In the method ofKapfhammer and Bischoff acetone was used on account of its selective solvent action on the reineckate, for the further purification of the precipitate after washing with ice-cold water and absolute alcohol. The acetone was then evaporated and after several extractions with ether the insoluble residue was dried and weighed. In the present method the reineckate is dissolved in acetone, and by comparing the colour of this acetone solution with that of a standard acetone solution of choline or acetylcholine reineckate, the amount of choline or acetylcholine combined as reineckate can be determined. The depth of the red colour depends on the amount of the reineckate radicle present. The colours are the same in equimolecular acetone solutions of choline and acetylcholine reineckates, since 1 mol. of each base combines with 1 mol. of reineckate. By this method quantities as small as 0-2 mg. of choline chloride and 0-32 mg. of acetylcholine bromide can be determined.