430 V. CHEMISTRY OF PHOSPHATIDES AND CEREBROSIDES 



tions are employed, choline gives a precipitate with potassium triiodide in 

 dilutions which vary between 1 : 20,000 and 1 : 2,000,000,^" depending upon 

 the conditions ^^*'^^'; precipitation occurs with the reineckate at 1:50,000, 

 while the phosphotungstate and phosphomolybdate are precipitated at dilu- 

 tions of approximately 1 : 20,000 and 1 : 10,000, respectively. ^^^ 



The most satisfactory microchemical determinations of choline in biologi- 

 cal material have been based upon the insolubility of the choline periodate 

 and of the choline reineckate. The original procedure for the use of the 

 periodate employed by Stanek^^^ was later modified to prevent the simul- 

 taneous precipitation of betaine by potassium triiodide""; this could be 

 avoided by carrying out the reaction in neutral or slightly alkaline solution, 

 under which condition choline alone was precipitated. Stanek"^ later 

 showed that proteins, peptones, purmes, alkaloids, trimethylamine, tri- 

 gonelline, stachydrine, and muscarine may also separate out under certain 

 specific conditions on treatment with potassium triiodide. Special modi- 

 fications of the Stanek method have been suggested by Sharpe"^ and later 

 by Roman. ^"^^ Further revisions of the Roman method have recently been 

 proposed. ^^'"^ 



The reineckate procedure has proved non-specific, although it is an excel- 

 lent and very sensitive method in the a])sence of interfering substances. 

 Other quaternary bases, tertiary and secondary amines, heterocychc com- 

 pounds as well as co-amino acids, all of which form insoluble reineckates, 

 may cause fallacious results. Thus, in the determination of acetylcholine 

 in muscle, the Kapfhammer and Bischoff procedure,"^ which involves the 

 use of reineckate, gave a value of 194 y per gram,"* while the values ob- 

 tained by either of the biological procedures"^- "^ did not exceed 0.08 7 per 

 gram. This discrepancy may be due in part to the failure to prevent the 

 rapid enzymic hydrolysis of acetylcholine; the procedure employed by 

 Bischoff et aW^ did not prevent this change. Strack and co-workers "^ 

 also, were unable to prove the presence of free choline in beef, dog, or rabbit 

 muscle. It was finally showii that the discrepancy between the reineckate 

 and the biological methods was to be ascribed to the fact that carnitine, 



i»7 T. Kinoshita, Arch. ges. Physiol. {Pfliiger's), 132, 607-631 (1910). 



i«8 F. J. Booth, Biochem. J., 29, 2064-2066 (1935). 



i«s E. Kahane and J. Levy, Bull. soc. chim. bioL, 21, 22.3-240 (1939). 



17" V. StaiiCk, Z. physiol. Chem., 47, 83-87 (1906). 



>" V. Stan6k, Z. physiol. Chem., 48, 334-346 (1906). 



1" J. S. Sharpe, Biochem. J., 17, 41-42 (1923). 



1" I. Reifer, New Zealand J. Sci. Tech., B22, 111-116 (1941). 



"* J. Kapfhammer and C. Bischoff, Z. physiol. Chem., 191, 179-182 (1930). 



"8 C. Bischoff, W. Grab, and J. Kapfhammer, Z. physiol. Chem., 207, 57-77 (1932). 



"6 F. Plattner and E. Krannich, Arch. ges. Physiol. (Pfluger's), 229, 730-737 (1932); 

 230 356-362 (1932). 



1" H. C. Chang and J. H. Gaddum, J. Physiol, 79, 255-285 (1933). 



"* E. Strack, P. Wordehoff, E. Neul)aur, and M. Gcissendorfer, Z. physiol. Chem., 

 233, 189-203 (1935). 



