VII. ESTIMATION 55 



gravimetric procedure for the assay of choline chloride in pharmaceutical 

 products, based on the precipitation of choline from absolute ethanol with 

 phosphotungstic acid, has been described by Gakenheimer and Reguera.^^ 

 A recent gravimetric method^^ consists of precipitating the cadmium chlo- 

 ride complex of choline from an alcoholic solution. This method can be 

 used for the assay of pharmaceutical products but has not been studied 

 with biological samples. 



B. MICROBIOLOGICAL PROCEDURES 



A microbiological method for the determination of choline by the use of 

 a mutant of Neiirospora crassa was described by Horowitz and Beadle.^^ 

 The mutant (34486) had been produced by ultraviolet irradiation of a 

 normal "wild type" strain. As a result of the induced mutation this organ- 

 ism fails to grow on an unsupplemented basal medium but grows if choline 

 is supplied. Combined choline, as in lecithin, is also utilized by the test 

 organism, but less efficiently than free choline. For this reason the choline 

 in samples must be liberated by hydrolysis prior to assay. Methionine has 

 a sparing action on choline and, when present in appreciable amounts, 

 must be removed from the sample. Jukes and Dornbush-^ have shown that 

 dimethylaminoethanol is also active for the mutant. 



A second cholineless strain (47904) which differs genetically from strain 

 34486, was described by Horowitz et aZ.,^°' ^^ and this mutant may also be 

 used in the bioassay of choline. The growth of both cholineless mutants is 

 stimulated by such compounds as acetylcholine, phosphorylcholine, mono- 

 methylaminoethanol and dimethylaminoethanol. The following compounds 

 were foinid to be inacti^'e for both mutants: betaine, creatine, sarcosine, 

 aminoethanol, neurine, diethylaminoethanol, dimethylamine, trimethyl- 

 amine, and tetramethylammonium chloride. 



The procedure for the Neurospora assay has been described in several 

 texts.-' ^' ^'- According to the original method^^ the sample for assay is 

 autoclaved with 3 % sulfuric acid for 2 hours at 15 pounds pressure to 

 liberate choline from its combined forms, and the solution is neutralized 

 with barium hydroxide. The solution is passed through a column containing 

 Permutit in order to separate the choline from methionine, and the ab- 



26 W. C. Gakenheimer ami R. M. Reguera, ,/. Aw. Pharm. /Issoc. Sci. Ed. 35, 311 



(1946). 

 "W. Seaman, J. J. Hugonet , and W. Leibmann, Anal. Chem. 21, 411 (1949). 



28 N. H. Horowitz and G. W. lieadle, ./. Biol Chem. 150, 325 (1943). 



29 T. H. Jukes and A. C. Dornhusli, Proc. Soc. Exptl. Biol Med. 58, 142 (1945). 



•'" X. H. Horowitz, D. Bonner, and M. B. Houlahan, J. Biol Chem. 159, 145 (1945). 

 31 X. H. Horowitz, ./. Biol Chem. 162, 413 (1946). 



^2 B. C. Johnson, Methods of Vitamin Determination, ]). 95. Burgess rublishing Co.. 

 Minneapolis, 1948. 



