ASSAY METHODS 67 



choline reineckate, however. 191 It is doubtful whether the method could 

 be used directly, unless by the Winzler and Meserve modification, for the 

 determination of choline in plants that contain substantial amounts of 

 alkaloids. 



Microbiological Assay. The microbiological method for choline which 

 has been applied to tissues, blood and urine is that originated by Horowitz 

 and Beadle, 192 and involves the use of a cholineless mutant of Neurospora. 

 This organism has lost by ultraviolet radiation its ability to synthesize 

 choline and gives corresponding growth responses when amounts of 

 choline, 1 fig to 50 ^g, are added to 25 ml of culture medium. The responses 

 are evaluated after 72 hours' growth by weighing the dried mycelia (ca. 

 5.0-50 mg). An alternate simplified procedure involves measuring the 

 diameter of the mold cultures as grown in 16 hours on agar in Petri 

 dishes. 193 - 194 



The response of this cholineless mutant as well as other cholineless 

 strains 194 is not quite specific. Betaine and ethanolamine are inactive, but 

 about 50 per cent of the choline of lecithins 192 is effective in the 72-hour 

 test; methionine is 0.2 per cent as active as choline; and dimethylamino- 

 ethanol and monoethylaminoethanol, acetylcholine, phosphorylcholine and 

 arsenocholine are highly active. Certain other synthetic homologues, 

 dimethylethylhydroxyethylamine, and diethylmethylhydroxyethylamine 

 are also highly active. 194 



One cannot help feeling that these nonspecificities are less important 

 than they appear. The authors are to be commended for thoroughness in 

 investigating the question of specificity, and the method should not be 

 damned because its specificity has been thoroughly investigated. Lecithins 

 and methionine can be eliminated from test solutions, and the other active 

 compounds for the most part are not known to occur naturally in quanti- 

 ties likely to interfere. Dimethylaminoethanol occurs in an alkaloid found 

 in certain leguminous plants 195 ; but its general distribution, while pos- 

 sible, and perhaps even probable, 196 has not been demonstrated. Leucke 

 and Pearson have applied the mold method to free choline in plasma and 

 urine, 197 and the free and bound choline of a few animal tissues, 198 with 

 results which appear to be satisfactory and in agreement with those 

 obtained by the reineckate method. Hodson has applied the method to 

 milk products. 163 A combination of the reineckate and microbiological 

 methods suggests itself as desirable in crucial cases. 



The basis for another microbiological test of limited interest is the 

 requirement of Type III Pneumococcus for choline. 199 A large number of 

 compounds, mostly synthetic, showed activity, including dimethylethanol- 

 amine (100 per cent) and ethanolamine (10 per cent). The nonspecificity 



