30 THE VITAMINS 



500-fold. If the final substance were in fact pure, then the concentration of this 

 substance in the original rice bran was presumably about 0.2 per cent, or in the 

 whole rice grain perhaps about 0.05 per cent. If, however, the final product were 

 still a mixture, then the percentage of the actual antineuritic substance in the rice 

 kernel must be smaller than this estimate would indicate. 



Suzuki and his coworkers also attempted by feeding experiments 

 to determine whether oryzanine plays as important a role with other 

 species as with pigeons and reported it to be indispensable for fowls, 

 mice, and dogs. In view of the later work of Cowgill, their experi- 

 ments with dogs are of particular interest. It was reported that dogs 

 when fed cooked rice and the boiled-out residue of horse flesh began to 

 lose appetite in about three weeks and after from five to seven weeks 

 showed marked losses in weight. If, however, 3 or 4 grams of the 

 alcoholic extract or 0.3 to 0.4 gram of Crude Oryzanine I were ad- 

 ministered daily to a dog which appeared about to die as a result of 

 the deficient diet, the appetite was quickly restored and the body 

 weight increased until the doses of oryzanine were omitted, when the 

 same symptoms recurred. In a full-grown dog these changes were 

 brought about four times in seven months. 



Vedder and Williams (1913) reported observations which raised 

 the question whether rice polishings contain antineuritic substances 

 other than the vitamin as apparently isolated by Funk or whether the 

 one substance is only partially separated by his method. 



They stated that before Funk's papers had reached them in the Philippines 

 they had attempted to concentrate the antineuritic constituent of rice polishings 

 by extraction with alcohol, precipitation with phosphotungstic acid, and decomposi- 

 tion of the phosphotungstate with barium hydroxide, but had not succeeded in 

 obtaining any protective action with the resulting material. In comparing methods, 

 they found that Funk had added 2.5 per cent of hydrochloric acid to the alcohol 

 and had used curative tests. On modifying their procedure by hydrolyzing the 

 alcoholic extract with 5 per cent hydrochloric or sulfuric acid they were able to 

 confirm his results in curative experiments. A comparison of the unhydrolyzed 

 and hydrolyzed extracts showed the former to be non-poisonous and only slowly 

 curative and the latter poisonous in larger doses and very promptly curative in 

 smaller doses. This difference was ascribed to the hydrolysis of the active material 

 into a more active and probably simpler form. On the theory that the marked 

 losses in activity of the phosphotungstate on decomposition with barium hydroxide 

 might be due to the destructive action of the barium hydroxide, the method was 

 xnodified by extracting the phosphotungstate precipitate by repeated prolonged 

 shaking with 50 per cent alcohol and adding barium hydroxide to the extract, 

 less being required than when added to the original precipitate. After removal 

 of the barium phosphotungstate the extract was treated with silver nitrate and 

 barium hydroxide as in Funk's method. 



On testing the various fractions they found that polyneuritis in fowls was 

 prevented not only by the fraction containing Funk's base, but also by the filtrate 

 from it and even by the "purine fraction" precipitated by silver nitrate from 

 neutral solution, if this fraction were fed in sufficient quantity. Of these three 

 groups of substances conferring protection, only one, the fraction containing 

 Funk's base, was promptly curative. 



