186 THE VITAMINES 



All these substances were administered to the pigeons intra- 

 muscularly, and from the results, it is evident that substance I had 

 the greatest therapeutic action. 



Others also investigated this question. Barsickow (487) concluded 

 that the therapeutic action was inherent only in the living or dead 

 yeast cells, and for the most part, he found the extracts inactive; 

 this has, of course, been shown to be incorrect. Edie, Evans, Moore, 

 Simpson and Webster (488) did not hydrolyze the yeast, but treated 

 the alcoholic extract, after the concentration of the solvent, directly 

 with silver nitrate and baryta. They obtained in this way a hygro- 

 scopic syrup which was very active for pigeons, 6 mg. being suffi- 

 cient, as a rule, for a cure. On further purification, a crystalline 

 substance was obtained which, because of the small yield, could not 

 be purified further. This crystalline substance was analyzed but the 

 data on its therapeutic action were lacking. 



Another method, useful for the purpose of removing a large mass of 

 inactive material, was described by us (489) in 1916. This method is 

 based upon the separation of the phosphotungstates by means of 

 the varying solubility in acetone. The residue, obtained from the 

 alcoholic extract of 100 kilos of dried yeast, was extracted with 10 

 per cent sulphuric acid, this extract-was then diluted half again with 

 water and precipitated with phosphotungstic acid. The resulting 

 precipitate, weighing 2237 grams was triturated several times with 

 dry acetone, leaving in the end 144 grams of insoluble material. This 

 portion, which represents 5.1 per cent of the total precipitate, con- 

 tained all the vitamine, according to our animal experiments. The 

 precipitate was decomposed with neutral lead acetate 2 and the 

 resulting solution precipitated with picric acid. The voluminous pre- 

 cipitate consisting of adenine picrate was filtered off. It was impos- 

 sible to crystallize the vitamine from the mother liquor, making it 

 necessary to continue the fractionation with sublimate, platinic 

 chloride, and picrolonic acid. The course of the fractionation, which 

 was controlled by animal experiments, is shown in the diagram 

 below, in which the double lines indicate the fraction containing the 

 vitamine. According to a patent by Bohringer and Sons (491) the 



2 We also attempted to decompose the precipitate by shaking it out with 

 amyl alcohol in acid solution according to Vah Slyke (490), but the method 

 was not quantitative because the last traces of phosphotungstic acid are 

 removable only with great difficulty. 



