502 V. CHEMISTRY OF PHOSP?IATIDES AND CEREBROSIDES 



even greater error was then introduced by omitting the zinc hydroxide pre- 

 cipitation on the hydrolyzed sample. Kirk,"*^^ by using a modification of 

 the second Kimmelstiel procedure, whereby the zinc hj'-droxide precipita- 

 tion was also used on the hydrolyzed sample, was able to obtain duplicates 

 within 4% when the cerebrosides were present to the extent of onlj^ 0.3 to 

 1.3 mg. 



Brand and Sperry^" introduced a new method which gives excellent 

 quantitative recoveries. Recognizing that the hydrolysis procedure of 

 Kirk is not sufficiently drastic to insure complete hydrolj'sis, the concen- 

 tration of the hydrochloric acid used was increased to 3 A'', and the reaction 

 was carried out in tightly stoppered flasks. Although Brand and Sperry 

 recognize the danger of destruction of the galactose when the acid concen- 

 trations are too high, no destruction occurred at the concentration of 3 A^ 

 over a 45-minute period. This sufficed for the complete hydrolysis of the 

 cerebroside. The determination of the galactose set free was made by the 

 eerie sulfate method of Miller and Van Slyke."*"^ 



Edman'*^^ suggested a new method based upon the colorimetric deter- 

 mination of the sugar by means of carbazole. Hydrolysis of the cerebro- 

 side is brought about with 2 A'' hydrochloric acid for 2 hours. Glycerol and 

 fatty acids must be removed before the reagent is added to determine 

 galactose. This micromethod can be applied to amounts of cerebroside as 

 low as 0.2 to 0.6 mg. Briickner^*" described a method for the separate 

 determination of the glucose- and galactose-containing cerebrosides by the 

 use of the orcinol test. 



Smith and Mair"*^^ have employed direct quantitative determination. 

 By this method a weighed amount of the dried brain is extracted in the 

 Soxhlet apparatus with hot chloroform; the chloroform residue is heated 

 with a methanolic baryta solution to saponify the phosphatides. After 

 addition of acetic acid it is evaporated to dryness; the residue is extracted 

 in a Soxhlet apparatus with hot acetone. The cerebroside separating on 

 cooling is filtered and weighed. 



9. Compounds Related to the Cerebrosides 



(1) Gangliosides 



In 1927, Walz,^^'^ working in Thierf elder's laboratory, isolated a cerebro- 

 side-like compound from beef brain and beef spleen which, however, dif- 

 fered from the kno\ra cerebrosides. It possessed a high sensitivity toward 



"" F. C. Brand and W. M. Sperry, J. Biol. Chem., I4I, 545-553 (1941). 

 ^'8 B. F. Miller and D. D. Van Slyke, /. Biol. Chem., II4, 583-595 (1936). 

 «» P. V. Edman, ./. Biol. Chem., US, 219-221 (1942). 

 "8" J. Bruckner, Z. phijsiol. Chem., 275, 73-79 (1942). 



^81 J. L. Smith and W. Mair, J. Path. Bad., 16, 131-135 (1911); 17, 123-126, 418-420 

 (1912). 



