II. CHEMISTRY 5 



effect of heating choline in aqueous baryta as well as in alcoholic solutions 

 of sodium ethylate and found only a negligible breakdown of choline after 

 boiling in baryta solution for 6 hours or after heating in a 5% sodium 

 alcoholate solution for 24 hours. 



Beattie^^ studied the acid hydrolysis of a lecithin emulsion prepared from 

 a commercial egg lecithin preparation and hydrolyzed in 7.8 % hydrochloric 

 acid at 110°. The maximum value of free choline was obtained after hy- 

 drolysis for 21 hours. Acid hydrolysis has been used also in the liberation 

 of choline from bound forms in tissues. ^°"-^ Ducet and Kahane^^ refluxed 

 animal and vegetable tissues with 30% nitric acid until a clear solution 

 was obtained. After neutralization of the solution with powdered calcium 

 carbonate and dilution with several volumes of water, 10 ml. of 50 % ferric 

 sulfate and 5 g. of calcium carbonate were added for each gram of dry 

 tissue originally taken. The mixture was heated to boiling and filtered. 

 The filtrate and washings containing the choline were concentrated to a 

 small volume, and the choline was precipitated by one of the reagents 

 generally employed for this purpose. These workers found that no choline 

 was destroyed during this procedure. 



The earliest methods which were employed for isolating choline from 

 biological extracts were dependent on the use of various sensitive though 

 non-specific precipitants. Choline may be precipitated from alcoholic solu- 

 tions as the double salt of platinum, gold, or mercury chlorides.^^- ^s. 24 

 Precipitation as the reineckate or the periodide has been employed most 

 extensively for the removal of choline from aqueous solution. 



Beattie^^ observed that a quantitative precipitation of free choline as the 

 reineckate can be obtained in solutions containing as little as 0.03 mg. of 

 choline chloride per milliliter and that the choline in about 7 to 10 ml. of 

 a solution of this concentration can be quantitatively determined. The 

 slight extent to which other substances interfere mth the reineckate pre- 

 cipitation and estimation of choline in animal tissues and fluids was demon- 

 strated by Beattie by analysis of tissue extracts, a tryptic digest, and urine 

 before and after the addition of known amounts of choline chloride (Ta- 

 ble I). 



16 B. N. Erickson, I. Avrin, D. M. Teague, and H. H. Williams, /. Biol. Chem. 135, 



671 (1940). 

 " H. P. Jacobi, C. A. Baumann, and W. J. Meek, J. Biol. Chem. 138, 571 (1941). 

 '^ J. Kapfhammer and C. Bischoff, Hoppe-Seyler's Z. physiol. Chem. 191, 179 (1930). 

 " F. J. R. Beattie, Biochem. J. 30, 1554 (1936). 



2« J. D. Fletcher, C. H. Best, and O. M. Solandt, Biochem. J. 29, 2278 (1935). 

 21 R. W. Luecke and P. B. Pearson, J. Biol. Chem. 153, 259 (1944); 155, 507 (1944). 

 " G. Ducet and E. Kahane, Bvll. soc. chim. biol. 28, 794 (1946). 

 " A. Lohmann, Arch. ges. Physiol. 122, 203 (1908). 

 " C. T. Morner, Hoppe-Seyler's Z. physiol. Chem. 22, 514 (1896). 



