450 V. CHEMISTRY OF PHOSPHATIDES AND CEREBROSIDES 



saponification was employed, which will not split the NH — CO linkage, 

 palmitic acid and unesterified lignocerylsphingomyelin were obtained. The 

 authors believe that sphingomyelin exists in both the esterified and the free 

 form in natui'o. Howp\tm\ the suggestion is made that it may occur only 

 in the form of fatty acid esters; the apj)ear;iiic(' of free sphingom^yelin 

 would then be the result of autolysis of the ester during the process of isola- 

 tion. A method is given by Thannhauser and Jieicheb^^" to determine the 

 proportion in the free and in the ester form by the ketene acetylation of the 

 ceramide. A value of 67.5% was reported for one sample. The formula 

 for the palmitic acid ester of lignocerylsphingomyelin would show the ac- 

 companying structure. Reichel and Thannhauser'''-'' have prepared a num- 

 ber of lignocerylsphingosine fatty acifl esters, which they call sphingosine 



c=o 



H /OH 



CH3(CH2),2Crt=CHC-C-CH20P=0 CH, 



H NH OCHjCHjN-CHj 



(CHo)^? 

 I 

 CH3 



Palmitic acid ester of lignoceo'lsphingomyelin 



fats. This demonstrates that the esterification of both hydroxyl groups in 

 sphingosine is possible. 



(2) Preparation of the Sphingoniyeliris 



a. Rosenheim and Tebb Method."^ Brain is used as the source of 

 sphingomyelin in this procedure. The cerebroside is first removed from 

 the brain tissue by extraction with cold pyridine. The sphingomyelin is 

 then extracted by allowing the brain to stand with 3 volumes of pyridine at 

 40-45°C. for one-half hour, after which it is filtered. The filtrate is cooled 

 to 15°C., and the precipitated sphingomyelin is filtered off and washed with 

 acetone. It is a white, ncn-hydroscopic powder, which is further purified 

 through repeated fractional precipitation from an alcchol-chloroform solu- 

 tion with acetone. It is finally crystallized from pyridine. 



b. Levene Method. -^'^ The dried brain sample is exhaustively ex- 

 tracted with boiling alcohol. The precipitate which forms on cooling the 

 alcohol extract is extracted with ether and acetone. The precipitate is then 

 taken up in hot pyridine, the crude sphingomyelin which separates on cool- 

 ing is filtered off, and the sphingomyelin is precipitated by acetone after 

 concentration of the acetic acid filtrate in vacuo to a small volume. The 

 crude preparation can be further purified by dissolving in a mixture of 5 



32« M. Reichel and S. J. Thannhauser, J. Biol. Chem., 135, 15-21 (1940). 



