246 CAROTINOIDS AND RELATED PIGMENTS 



that the chemistry of the blue stain with Nile blue argues against 

 this supposition. As stated by Smith the simultaneous staining of 

 fatty acids and neutral fat by Nile blue is due to the fact that this 

 dye is a mixure of a strongly basic oxazine which reacts readily with 

 fatty acids to form blue soaps, and a weakly basic oxazone which 

 dissolves readily in neutral fats and fat solvents. When it is remem- 

 bered that none of the carotinoids have acid properties it may be 

 argued that the blue stain imparted to carotinoid pigment granules in 

 the writer's and in Dolley and Guthrie's experiments merely indi- 

 cates the acid character of the lipoid with which the pigment was as- 

 sociated. This argument would have to be accepted as conclusive if 

 the oxidized pigment granules should be found to retain their prop- 

 erty of taking the blue stain with the Nile blue oxazine like they have 

 been observed to do with Sudan III and Scarlet Red. On the other 

 hand, if it should be found that the oxidized pigment granules in 

 plant or animal tissues no longer take the blue stain with Nile blue 

 there would be strong basis for believing that the oxazine base in the 

 dye is specific for carotinoids as well as for fatty acids. Certainly 

 it would not be unreasonable to assume that the profound changes 

 which undoubtedly occur in the chemical characters of the carotinoids 

 during their oxidation also alter their relation towards dyes. 



Hueck (1912) has stated that lipochrome in animal tissue still 

 stains blue with Nile blue after oxidation with hydrogen peroxide. 

 At the writer's suggestion Dr. Dolley has investigated this point more 

 exhaustively using frozen sections of carrot tissue, with the result 2 

 that Hueck's observation is confirmed. Complete oxidation with 

 hydrogen peroxide and sunlight or careful oxidation with ferric chloride 

 fails to destroy the ability of the visible, bleached pigment granules to 

 take the blue oxazine base from the Nile blue dye. It thus appears 

 impossible to differentiate between carotinoid pigment and fatty acids, 

 although these can be distinguished from neutral fat or esters by the 

 Nile blue dye. 



The effect of ferric chloride on the carotinoids is alone of some 

 value in indicating the presence of carotinoids in animal tissues. 

 While it is not possible to obtain the green color reaction when work- 

 ing with tissue sections, on account of the fact that the color reaction 

 is in the reagent itself, pigment granules which are readily oxidized 

 (bleached) by this reagent may be suspected to be carotinoid in na- 

 ture. Dolley and Guthrie found that a strong solution of ferric 



2 Personal communication. 



