480 V. CHEMISTRY OF PHOSPHATIDES AND CEREBROSIDES 



tates at 37 °C. is removed, and the precipitate of cerasine which forms at 

 room temperature is removed. This purification may be repeated. The 

 final purification involves recrystallization from a large amount of 90% 

 acetone containing 2% pyridine. On an average, only 1.36 g. of pure 

 material is obtained from 10 g. of cmde cerasine. 



(3) Loening and Thierfelder Method for Removing Phosphatides by Use of 



Barium Hydroxide^-^ 



The most difficult procedure in the purification of the cerebrosides is the 

 removal of the phosphatide impurities. I^oening and Thierfelder have 

 accomplished this l)y hydrolysis of these compounds with saturated barium 

 hydroxide; this procedure leaves the cerebroside unaltered. 



Saturated barium hydroxide is added to 30 g. of the "protagon" obtained 

 by the Thierfelder method (Method 1), and the mixture is reduced to a fine 

 emulsion which is free from large particles. The volume is made up to 1100 

 ml. and the mixture is heated in a boiling water bath for 70 minutes, with 

 constant agitation. After cooling, the precipitate is filtered and washed with 

 water to remove the barium and then with acetone to remove the water. 

 The residue is ground with acetone and is then heated at boiling tempera- 

 ture with 1000 ml. of acetone for 3 minutes with shaking. It is then filtered 

 through a hot-water funnel. After cooling in a refrigerator, the precipitate 

 is filtered and the filtrate is used for further extraction. The digestion of 

 the precipitate with acetone is repeated a number of times, first for only a 

 few minutes, then for 15 minutes, for 30 minutes, and finally for a number of 

 hours. The first precipitates are phosphorus-free, while the later ones con- 

 tain small amounts of phosphorus. Additional phosphorus-free material 

 can be obtained from the impure precipitates by a new extraction with ace- 

 tone. About 25 g. of the partially purified cerebrosides is dissolved in 70 

 volumes of absolute alcohol, and the solution is allowed to stand overnight 

 at 29 °C. The precipitate is removed by filtration through a hot-water 

 funnel. The precipitate is cerebroside and accounts for about 70% of the 

 total cerebroside. It consists chiefly of phrenosine. The further purifica- 

 tion and separation of the cerebrosides into phrenosine and cerasine can be 

 carried out from this point as described earlier. 



(4) Preparation of Nervone According to Method of Klenk'^" 



a. Separation of Crude Nervone. The brain is macerated, dehydrated, 

 and prepared for extraction according to the Thierfelder method. '*^^'^^'' A 

 petroleum ether extract of 10 human brains is concentrated to 3 liters and 

 refrigerated overnight. The precipitated protagon is removed, the filtrate 

 is reduced to 500 ml., and 2.5 liters of absolute alcohol is slowly added, with 

 continual stirring. The clear supernatant fluid is poured off from the sticky 



■'^'i H. Loening and PI. Thierfelder, Z. physiol. Chem., 74, 282-289 (1911). 



