THE SPHINGOMYELINS 457 



parts of ligroin and 1 part of alcohol, ^^^ after which alcohol is added as long 

 as a precipitate forms. This is filtered, and the filtrate is allowed to stand 

 overnight at 0°C. The solution is filtered, concentrated under reduced 

 pressure, and the sphingomyelin is precipitated by the addition of acetone. 

 Final purifications are made by crystallization from a mixture of equal parts 

 of pyridine and chloroform, first at room temperature, then at 30°C., and 

 finally at 37°C. Such a preparation has been shown to be free from cere- 

 brosides. 



c. Klenk and Rennkamp MethodJ^^ A somewhat different procedure 

 has been proposed by Klenk and Rennkamp which yields a glycerol-free 

 sphingomyelin preparation. "Crude dry carbohydrate-free sphingomyelin 

 is dissolved in 10 volumes of benzene (b.p., 70-80°C.) and treated with 1 

 volume of sodium ethylate in ethyl alcohol prepared by dissolving 1 g. of 

 sodium in 100 cc. of ethyl alcohol. Transesterification of glycerides occurs 

 during 1 hour of refluxing. The solvents are evaporated and the esters are 

 removed with acetone; the sphingomyelin is freed from glycerophosphoric 

 acid by precipitation by cadmium acetate from methanol and adsorption of 

 the last trace by AI2O3. The final crystallization materials had a [q:]d = 

 + 5.30-5.35 (0.54 g. in 10 cc. methanol-CHClg, 1 : 1)." 



d. Thannhauser, Benotti, and Boncoddo Method. ^^^ Since all the 

 methods used for the preparation of sphingomyelin result in a product 

 which is a mixture of sphingomyelin and hydrolecithin in various propor- 

 tions, the present procedures are designed to prepare lung sphingomj^elin 

 free from hydrolecithin. The difficulty in separating the two products re- 

 sults from the similarity of their physical properties, especially since both 

 substances are insoluble in ether. 



(a) Method 1 . The ether-soluble lipids are first remo\'ed from macerated 

 lung tissue which has previously been washed twice with acetone and dried 

 in vacuo at 60°C., by exhaustive extraction of the powdered material for 3 

 days in a continuous extractor with ether. -^^ The crude sphingomyelin is 

 extracted with glacial acetic acid, precipitated with acetone, filtered and 

 dried after washing with acetone. This material is free from unsaturated 

 monophosphatides, but contains hydrolecithin to the extent of approxi- 

 mately 33%. 



To remove the hydrolecithin, 10 g. of the crude phosphatide is suspended 

 in a small amount of water and ground to a paste; 200 ml. of 0.25 A^ NaOH 

 is added. ^^^ This suspension is then shaken for 5 days at 37°C. After 

 acidification with glacial acetic acid, it is cooled in the refrigerator overnight 

 and filtered with the aid of a Hyflo filter. The precipitate is washed with 

 acetone and then extracted for 2 or 3 days in a Soxhlet apparatus, to remove 

 any free iaXiy acids. The precipitate is dialyzed for 24 hours against run- 

 ning water, to remove inorganic contaminants. The dialyzed suspension is 



'21 E. Klenk and F. Rennkamp, Z. physiol. Chem., 267, 1-45-15:3 (1940-1941). 



