4 CHOLINE 



since these original isolations. Wrede and Bruch^ extracted various tissues 

 mth hot acidulated water, and the choline in these extracts was isolated 

 and weighed as the chloroaurate. Bischoff et al.,^ using a reineckate precipi- 

 tation, reported finding up to 45 mg. of free choline (calculated as the 

 chloride) per kilogram of muscle. Heesch^ prepared extracts of blood serum 

 which had been treated with trichloroacetic acid and found in these ex- 

 tracts 2.5 to 10 mg. of choline per liter of blood. Strack et aZ.^" have pre- 

 sented data which suggest that much of the evidence for the presence of 

 free choline in biological materials is unreliable, owing to delay in the 

 preparation of extracts, with resulting release of choline by autolysis. They 

 found that dog's liver contained to 43 mg. of choline if extracted imme- 

 diately after death of the animal and 136 to 164 mg. of choline per kilo- 

 gram of liver if extracts were made 5 hours after death. A similar slow 

 release of free choline occurred in experiments in which the fresh tissue 

 was suspended in alcohol. Strack et alM did not find free choline in rabbit, 

 dog, or beef muscle. On careful investigation the substance in muscle which 

 was precipitated as the reineckate and reported as choline by Bischoff et 

 al.^ was found to be carnitine. 



Many solvents have been tested with respect to the thoroughness with 

 which total choline, combined and free, is extracted from natural products. 

 Among these are benzene, petroleum ether, ethyl ether, ethanol, methanol, 

 acetone, chloroform, and mixtures thereof. None has proved to have any 

 special advantage over methanol itself .i^- ^^ EngeP- employed multiple ex- 

 tractions of samples with methanol in a Bailey- Walker extractor. The more 

 convenient method of extraction with the Soxhlet apparatus is generally 

 preferred. Glick^^ has recommended the mixing of powdered samples with 

 No. 2 pulverized pumice, after weighing, to prevent caking of the sample 

 and the resultant channeling of the extracting solvent. 



The residue of the methanol extracts of samples must be hydrolyzed if 

 the total choline content is to be determined. Barium hydroxide has been 

 favored as the alkali for the digestion process because there is no loss of 

 choline when pure choline solutions are used.^^"^^ Gulewitsch^^ studied the 



7 F. Wrede and E. Bruch, Hoppe-Seyler's Z. physiol. Chem. 195, 255 (1931). 



8 G. Bischoff, W. Grab, and J. Kapfhammer, Hoppe-Seyler's Z. physiol. Chem. 207, 

 57 (1932). 



9 0. Heesch, Arch. ges. Physiol. 209, 779 (1925). 



10 E. Strack, E. Neubaur, and H. Geissendorfer, Hoppe-Seyler's Z. physiol. Chem. 



220, 217 (1933). 

 " E. Strack, P. Wordchoff, E. Neubaur, and H. Geissendorfer, Hoppe-Seyler's Z. 



phxjsiol. Chem. 233, 189 (1935). 



12 R. W. Engel, J. Biol. Chem. 144, 701 (1942). 



13 M. Rhian, R. J. Evans, and J. L. St. John, /. Nutrition 25, 1 (1943). 

 " D. Glick, J. Biol. Chem. 156, 643 (1944). 



15 W. Gulewitsch, Hoppe-Seyler's Z. physiol. Chem. 24, 513 (1898). 



