66 THE BIOCHEMISTRY OF B VITAMINS 



levels at which inositol is effective in the test is from 10 to 30 fig, as com- 

 pared with 0.1 to 0.8 fig in the yeast tests. 



Biological Test Using Mice. The ability to cure alopecia in mice which 

 have been fed inositol-free diets was used as an assay method by Wool- 

 ley, 179 but the results obtained were only roughly quantitative, partly 

 because relatively few levels were tested. Phytin and mesoinositol, for 

 example, were found to be "active" when tested at the same level m (0.1 

 per cent of the diet) . If the active ingredient is inositol, phytin should 

 be about 1/5 as active. The relative activities of phytin and inositol for 

 mice would throw some light on the assimilation of phytin and its effects 

 on calcium assimilation. 



Choline 



Chemical, microbiological, physiological and animal assay methods for 

 choline have been described. Each has its usefulness, though the methods 

 in general are not as satisfactory as many that have been described in 

 previous sections. 



Chemical Assay. A large number of chemical tests have been applied 

 to the problem of the estimation of choline. 180 Among these precipitation 

 with reineckate is without question the most valuable. The use of this 

 method has a long history which we shall not take time to trace. Among 

 more recent papers those by Beattie, 181 Jacobi and co-workers, 182 Engel, 183 

 Marenzi and Cardini, 181 Enteman et al. 1S5 Glick, 186 and Winzler and 

 Meserve, 187 appear to be most important. The subject has been reviewed 

 recently by Handler. 188 



In most cases choline is freed by alkaline hydrolysis, and choline 

 reineckate precipitated under prescribed conditions is determined colori- 

 metrically in acetone solution. Marenzi and Cardini, 184 however, deter- 

 mined chromium colorimetrically in the insoluble reineckate, and Winzler 

 and Meserve 185 used ultraviolet light instead of visible light. 



This general method has been applied to tissues and feeds most exten- 

 sively by Engel 189 and Rhian and co-workers, 190 with results which 

 appear satisfactory. None of the methods is extremely sensitive. For the 

 direct colorimetric determination of the reineckate in acetone about 200 

 fig of choline must be in the sample ; when ultraviolet light is used 185 the 

 minimum determinable amount is about 50 fig. Colorimetric analysis for 

 chromium 1S4 makes possible a determination of 15 fig. As long as one is 

 concerned with the total choline content of tissues, which is relatively 

 high, the methods are serviceable from the standpoint of sensitivity. 



Means have been devised whereby most of the interfering substances 

 commonly found in food materials, including betaine, are eliminated. 

 Dimethyl aminoethanol may be carried down to some extent along with 



