54 CHOLINE 



ether mixture and the residue of this extract was saponified for 2 hours at 

 80° with baryta. The choHne, precipitated as the reineckate, was dissolved 

 in acetone and the color intensity was measured, using a light filter which 

 transmits at 520 m/x. 



EngeP^ advised more exhaustive extraction of biological samples with 

 methanol and hydrolysis of extracts with baryta for 2 hours at 100° rather 

 than at 80°, as recommended by Jacobi et al. Glick'^ further improved the 

 reineckate method by showing that interference due to many compounds 

 which form insoluble reineckates is circumvented by precipitating the 

 choline reineckate in an alkaline medium. Propanol was found to be the 

 most suitable solvent for washing the reineckate. The maximum light 

 absorption for choline reineckate, as determined spectrophotometrically by 

 Glick, is near 526 m^u. Click's reineckate method and the more recently 

 published modification by Willstaedt et al." are in wide use today. 



The development of a brown color, by adding an iodine reagent to an 

 aqueous solution of choline reineckate, was used as the basis of a method 

 by Shaw.i* Still another modification of the reineckate method was proposed 

 by Marenzi and Cardini.^^ It is based on the oxidation of the chromium of 

 choline reineckate to the chromic state by means of alkaline hydrogen 

 peroxide, followed by colorimetric determination of the chromate by means 

 of the violet-red color produced in an acid solution with diphenylcarbazide 

 (Cazeneuve's reaction). The authors claim that this method will allow 

 the estimation of as little as 15 j of choline. Winzler and Meserve^" in- 

 creased the sensitivity of the reineckate method by making use of the very 

 great absorption of acetone solutions of choline reineckate at 327 m/u. This 

 method has an accuracy of ±5 % with samples containing 50 to 400 y ^^ 

 choline hydrochloride. 



Lintzell and Fomin'^ introduced a new micromethod for choline estima- 

 tion based on the oxidative degradation of choline to trimethylamine and 

 titration of the latter. Neither the original method nor the modifications 

 of if--' -^ appears to have gained general acceptance. 



Less well-known methods for the determination of choline are those of 

 Ambo and Aoki-^ and of Eagle,'-^ who based their procedures on the ^'olu- 

 metric determination of mercury in the mercuric salt of choline. A simple 



" H. Willstaedt, M. Borggard, and H. Lieck, Z. Vitaminforsch. 18, 25 (1946). 



18 F. H. Shaw, Biochem. J. 32, 1002 (1938). 



19 A. D. Marenzi and C. E. Cardini, J. Biol. Chem. 147, .363 (1943), 



20 R. J. Winzler and E. R. Meserve, J. Biol. Chem. 159, 395 (1945). 



21 W. Lintzel and S. Fomin, Biochem. Z. 238, 438 (1931). 



22 G. Klein and H. Linser, Biochem. Z. 250, 220 (1932). 



23 W. Lintzel and G. Monasterio, Biochem. Z. 241, 273 (1931). 



2-4 H. Ambo and T. Aoki, Trans. Japan. Pathol. Soc. 21, 171 (1931). 

 « E. Eagle, J. Lab. Clin. Med. 27, 103 (1941). 



