METHODS OF ISOLATION OF CAROTINOIDS 207 



to separate when the clot contracts, or by defibrinating the freshly 

 drawn blood by whipping it vigorously, filtering off the fibrin and 

 ci'titrifuging the erythrocytcs from the defibrinatcd plasma, or merely 

 by drawing the blood into sufficient saturated potassium oxalate or 

 sodium citrate solution to prevent clotting and throwing down the 

 erythrocytes from the oxalated or citrated blood with the centrifuge. 

 Each of the three preparations, namely, serum, defibrinated plasma 

 or oxalated (or citrated) plasma serve equally well for the isolation 

 of the serum carotinoids. 



In most cases carotin, when present in blood, appears to be in 

 some sort of physico-chemical combination with a fraction of the 

 albumin in colloidal solution in the blood. Whatever the expla- 

 nation of the state of the pigment in the blood may be in these 

 cases, the fact remains that when this occurs the direct extraction 

 of the pigment with ether, petroleum ether, chloroform, carbon disul- 

 fide or any of the usual carotin solvents is impossible. However, if 

 the serum is first treated with an equal volume of alcohol, the 

 carotin can be readily extracted by shaking with the solvents men- 

 tioned. Based on this fact the writer devised the following method 

 for extracting the carotin from blood serum: Clear serum or plasma 

 is mixed with an excess of plaster of Paris, using about 40 grams 

 of the CaS0 4 for each 10 cc. of serum. The damp powder is trans- 

 ferred to a flask, alcohol added equal to the volume of serum and 

 thoroughly mixed with the plaster of Paris mass. An equal volume 

 of low boiling petroleum ether is now added and vigorously shaken 

 with the mass. On standing, the petroleum ether rises to the surface, 

 giving an almost quantitative extraction of the carotin. The extract 

 can be readily poured off and the extraction repeated with fresh 

 petroleum ether in order to insure a complete extraction. 



Reference has already been made to the manner in which blood 

 carries the carotin. Until recently the writer held the view that 

 carotin is always present in some sort of combination with an albumin 

 fraction in the serum. So far as his experience with the blood of 

 cattle and horses is concerned this view still holds. However, he has 

 recently examined the blood of several diabetics on vegetarian diets 

 containing much green food in which this carotin-albumin com- 

 bination did not appear to exist. At least the pigment, which proved 

 to be carotin, or at any rate to have the relative solubility and other 

 chemical properties of carotin, and not xanthophyll, was readily and 

 completely extracted from the serum merely by vigorous shaking with 



