214 CAROTINOIDS AND RELATED PIGMENTS 



at boiling temperature for about one hour, taking care to use alco- 

 holic potash which itself gives rise to no color on heating. The 

 pigment is extracted from the saponified material using the pro- 

 cedure given for isolating the pigment of butter fat. The ether solu- 

 tion of pigment is dried by filtering through a layer of anhydrous 

 Na 2 S0 4 and evaporated to dryness in vacuum. The chief impurity 

 in the residue will be cholesterol. By dissolving in the least possible 

 amount of hot methyl alcohol and cooling to a low temperature a 

 great deal of the cholesterol will precipitate out and can be removed 

 by filtration. The cholesterol which remains will not interfere with 

 the examination of the pigment. In the writer's experience egg yolk 

 pigment prepared in this way will invariably show the presence of 

 a small amount of pigment which cannot be extracted from petro- 

 leum ether by 80 per cent methyl alcohol, indicating that carotin-like 

 pigments are not entirely absent from egg yolk. 



Blood serum. It is not necessary to dwell at length on the isola- 

 tion of xanthophyll from blood serum in view of the detailed descrip- 

 tion already given of the procedure to be used for isolating carotin 

 from blood. One or two points, however, should be emphasized. 

 Xanthophylls are found most abundantly in the blood serum of fowls, 

 as has already been pointed out. This does not mean, however, that 

 blood rich in carotin, like cattle blood, is necessarily devoid of xantho- 

 phylls. In order to show the presence of these pigments in cattle 

 blood it is necessary to extract 200-300 cc. of desiccated (with CaS0 4 ) 

 serum completely with ether as well as with petroleum ether, after 

 treating with alcohol. The combined pigments from well colored 

 serum will show the presence of xanthophylls when submitted to the 

 phase test or analyzed by means of the chromatograph. 



It has been the writer's invariable experience with the blood of fowls 

 that the xanthophylls present can be readily extracted by direct shak- 

 ing of either the fresh or desiccated serum with ether. The experience 

 of van den Bergh and Muller (1920) has been contrary to this, these 

 investigators finding a number of cases in which ether extraction 

 failed. No explanation is as yet apparent for this divergence in our 

 experiences. However, in view of the fact that it appears possible 

 for cases to occur in which ether extraction alone fails to remove the 

 pigment the writer advises that desiccated blood serum, in which 

 xanthophylls are suspected to exist, be extracted first with ether, 

 then treated with alcohol and the ether extraction repeated. 



Until recently the writer believed that the direct extraction of caro- 



