412 V. CHEMISTRY OF PHOSPHATIDES AND CEREBROSIDES 



and is finally suspended in acetone to remove the toluene. The final 

 purification involves its precipitation as the cadmium salt, after which the 

 cadmium is removed and the lecithin is recrystallized. Levene and Rolf 

 obtained 80-100 g. of the pure cadmium salt from 11 kg. of dried egg-yolk. 

 Lecithin has also been prepared from liver^" and brain^^ by the application 

 of this method. Levene and Rolf/^ in later work, used a cold saturated 

 methyl alcohol solution of cadmium chloride for the precipitation of egg- 

 yolk lecithin. In the preparation of this phosphatide from liver, the 

 lecithin is extracted from the diced, finely macerated liver by alcohol 

 (28 liters for 20 kg.). The extract is then concentrated to one-third its 

 volume and is allowed to stand at 0°C. overnight. After filtration of the 

 precipitate, the lecithin is precipitated by methyl alcoholic cadmium 

 chloride solution. Li the case of brain, Levene and Rolf*^ employ two 

 preliminary acetone extractions to remove the bulk of contaminants (16 

 liters each for 20 kg. of hashed brain previously dried under a vacuum). 

 Following this treatment, the lecithin is extracted with 24 liters of alcohol. 

 The subsequent treatment from this point on is the same. The method of 

 Levene and Rolf for the preparation of lecithin has been somewhat simpli- 

 fied by Pangborn.^2 



{/i) Separation of a- and ^-Lecithins 



Methods for the separation of the a- and /8-forms of lecithins and ceph- 

 alins have been worJced out by Suzuki, Yokoyama, and Nishimoto,^''"^^ 

 and these have been modified by Welch. ^^ The microestimation method of 

 Welc})^^ involves the separation of the phospholipids from the tissues by 

 Bloor's procedure, ^^ with the exception that the alcoholic extract is con- 

 centrated in vacuo. 



The ether solution of the phospholipids is allowed to stand overnight in 

 the refrigerator, and the insoluble material is then removed by centrifuga- 

 tion. The phospholipids are reprecipitated from ether by acetone, to 

 remove any adhering fat and cholesterol. The lecithin and cephalin are 

 generally separated by treatment of the residue obtained on evaporation 

 of the ether extract with cold absolute ethyl alcohol and by allowing the 

 solution to remain in the refrigerator overnight. This is then centrifuged 

 cold and the supernatant is drawn off. The residue is again mixed with 

 more absolute alcohol, boiled, chilled, and centrifuged cold. The absolute 



60 p. A. Levene aud T. Ingvaldsen, ./. Biol Chern., /,% 359-378 (1920). 

 " P. A. Levene and L P. Rolf, /. Biol. Chem., 46, 353-365 (1921). 

 62 M. C. Pangborn, J. Biol. Chem., 137, 545-548 (1941). 

 6' B. Suzuki and Y. Yokoyama, Proc. Imp. Acad. Japan, 6, 341-344 (1930). 

 6* Y. Yokoyama and B. Suzuki, Proc. Imp. Acad. Japan, 8, 183-185 (1932). 

 6s U. Nishimoto and B. Suzuki, Proc. Imp. Acad. Japan, 8, 424-427 (1932). 

 66 U. Nishimoto, Proc. Imp. Acad. Japan, 10, 578-581 (1934). 

 " W. R. Bloor, /. Biol. Chem., 77, 53-73 (1928). 



