THE ANTIRACHITIC VITAMINE 221 



in petroleum ether is hydrolyzed with alcoholic potassium hydroxide 

 at ordinary temperature, and the soap solution neutralized, the active 

 substance may be concentrated in olive oil by extracting with the 

 latter. Osborne and Mendel (581) dissolved ox fat and butter in 

 alcohol at 40C. and allowed it to crystallize out on freezing at 

 20C. The A-vitamine could not be demonstrated in the crystal- 

 lized portion, but was found in the oily, uncrystallizable portion. 

 Drummond (582) attempted to repeat the work of McCollum and 

 Davis on the stability after saponification. After butter had been 

 saponified at the usual temperature, the solvent was removed in 

 vacuum, the soaps dissolved in water and the unsaponified portion 

 extracted with ether. All of the fractions thus obtained were tested 

 on rats and found inactive. Recently, Steenbock, Sell and Buell 

 (582a) repeated the saponification experiments and have found 

 that cod liver oil can be treated with 20 per cent alcoholic potassium 

 hydroxide solution for 4 hours at 37C. without losing its potency. 

 Since the active substance after dilution with water can be extracted 

 with ether, it was concluded that vitamine A is neither a fat nor 

 an ester. 



Numerous experiments were carried out for the purpose of con- 

 centrating this vitamine from various vegetables and grasses by 

 extraction. McCollum, Simmonds and Pitz (583) thought that the 

 vitamine could not be removed by extracting plant tissue with ether, 

 because it is in all probability bound to the protein. Still, Osborne 

 and Mendel (584) were able to do this. They dried spinach and 

 clover in a stream of air at 60C., and then extracted it with ether, 

 obtaining a very active substance. These results were confirmed by 

 Zilva (585) who obtained an active alcoholic and ether extract from 

 carrots. Steenbock and Boutwell (586) were unable to obtain an 

 active substance by extraction of carrots with ether alone, but could 

 do so with carbon disulphide, chloroform and benzol. In spite of 

 this, the residues showed almost the original activity. Using alfalfa 

 grass as a source, a simple ether extraction gave no result, while a 

 mixture of alcohol and benzol gave almost quantitative extraction. 

 The same workers then tried to make a fractionation by means of 

 solubility. Alfalfa was treated with cold alcohol, the latter distilled 

 off on the water bath and the residue allowed to stand over night 

 with 20 per cent alcoholic potassium hydroxide. When the saponifi- 

 cation had been treated with ether and filtered from the ether- 



