VITAMIN B {B^) 31 



In their attempts to discover a better method for isolating the 

 base, Vedder and WilHams also tried the method of Suzuki, Shima- 

 mura and Odake but do not seem to have regarded it as constituting 

 any marked improvement over the method of Funk. The method noted 

 above was also abandoned as a quantitative procedure as the greater 

 part of the curative base vi^as lost during the final precipitation with 

 silver nitrate in the presence of barium hydroxide. A method which 

 proved more successful consisted in neutralizing a quantity of unhy- 

 drolyzed extract of rice polishings with barium hydroxide, adding an 

 excess of barium acetate, treating the precipitate thus obtained with 

 5 per cent sulfuric acid for three hours and filtering. The filtrate after 

 the removal of the excess sulfuric acid with barium carbonate proved 

 to have retained practically all of the curative power of the original 

 extract as shown by curative experiments with fowls suffering from 

 advanced polyneuritis. 



In the same year. Cooper (1913a) described the preparation from 

 animal tissue (horse flesh) of a substance capable of curing polyneu- 

 ritis in birds. 



In Cooper's method, absolute alcohol followed by ordinary alcohol was used 

 to extract the active substance from the dried powdered flesh, ether to remove 

 lipoidal material from the extract, partial crystallization of an aqueous solution 

 of the alcoholic extract to remove carnosine, and lead acetate to remove other 

 impurities. The precipitation of the active material was effected first by silver 

 nitrate, and then by silver nitrate with barium hydroxide. It was estimated that 

 at least three-fifths of the total amount of antineuritic substance present in the 

 filtrate from the lead acetate precipitation was precipitated by the addition of 

 silver nitrate only, and about one-fourth of the remainder was carried out of 

 solution by the subsequent addition of baryta. The remaining three-twentieths 

 had probably decomposed under the influence of the alkali. The residue obtained 

 by evaporation of the curative solution resulting from dissolving the first silver 

 nitrate precipitate in hydrochloric acid was next extracted with chloroform. Only 

 a small amount of inactive substance was extracted. The active substance was 

 thus insoluble in chloroform and also insoluble in benzene and in ethyl acetate. 

 It was destroyed by standing in ammoniacal solution, and was not destroyed by 

 hydrogen sulfide but appeared to be largely lost by adsorption on precipitates of 

 metallic sulfides. 



Cooper also (1914b) made use of acetone precipitation in the 

 preparation of an antineuritic concentrate from cardiac muscle ; and 

 in that year made the first use of autolyzed yeast as a starting point 

 for the isolation of the antineuritic vitamin (1914). 



In a paper published in June 1913, Funk (1913b) gave the results 

 of further studies upon the chemistry of the vitamin fraction of yeast 

 and rice polishings. By recrystallizing from alcohol the active frac- 

 tion of yeast prepared by his first method he obtained three substances, 

 one of which had the composition C24H19O9N5 and one C29H23O9N5, 



