HYDROLYSIS PRODTTCTS OF CEREBROSIDES 497 



slightly soluble in cold ether (Twitchell separation) but more soluble in 

 warm ether. Nervonic acid cannot be precipitated from alcoholic solution 

 by magnesium. 



d. Hydroxynervonic Acid, (a) Structure. The conversion of hydroxyner- 

 vonic to cerebronic acid by hydrogenation indicates that it is an a-hydroxy- 

 acid of the C24 series. Inasmuch as, on decomposition with ozone, it yields 

 pelargonic acid, the double bond must be present between carbons 15 and 

 10. The structure which is assigned to it is CH3(CH2)7CH:CH(CH2)i2- 

 CHOHCOOH. 



(6) Properties. Klenk^**^ first prepared hydroxynervonic acid from a 

 cerebroside fraction partially soluble in petroleum ether. It is a white 

 crystalline solid, readily soluble in diethyl ether, chloroform, alcohol, and 

 acetone. It can be crystallized in needles (mostly in clusters) from 75% 

 alcohol. It melts at 65°C. 



Since the a-carbon atom is asymmetric, hydroxynervonic acid is optically 

 active. It is similar to cerebronic acid in showing dextro-rotation in pyri- 

 dine ([«]d° +2.87°),^^^ and levo-rotation in chloroform. ''^^ The iodine 

 number is 66.4. 



Sodium hydroxynervonate dissolves only in warm methyl or ethyl alco- 

 hols. The salt also dissolves in warm water. On cooling, it separates as a 

 voluminous precipitate. The acid is precipitated quantitatively from hot 

 alcoholic solution by magnesium acetate. Lead hydroxynervonate is 

 insoluble in hot ether. 



e. w-Hexacosenic Acid. A third unsaturated acid, w-hexacosenic acid, 

 has recently been isolated from the hydrolysate of brain cerebroside by 

 Klenk and Schumann. ^^^ These workers believe it to be one of the acids 

 normally present in cerebroside. The empirical formula is C26H50O2, and it 

 possesses one unsaturated linkage. The melting point is 45^5. 5°C., and 

 the iodine number of the preparations is approximately theoretical (64.3). 



n-Hexacosenic acid has likewise been reported in sphingomyelin. Al- 

 though it was present to the extent of only 3% in the cerebroside fatty 

 acids, it is not believed that this can be ascribed to an impurity in the 

 sphingomj'elin. 



f. Methods for Separation of the Acids. Klenk*^*'*^^ has proposed 

 several methods for the separation of the fatty acids from the hydrolysis 

 products of cerebrosides. If reliable results are to be obtained, it is neces- 

 sary that the cerebroside which is being analyzed be in as pure a state as 

 possible, and especially that it be free from phosphatides. 



After the cerebroside is hydrolyzed by boiling with methyl alcohol con- 

 taining sulfuric acid, the fatty acids precipitate on cooling in a refrigerator. 

 These are separated and washed with ice-cold methyl alcohol. They con- 

 sist chiefly of the methyl esters of the saturated fatty acids. 



*" E. Klenk and E. Schumann, Z. physiol. Chem., 272, 177-188 (1941-1942). 



