304 THE VITAMINS 



"cod liver oil mixed with ground grains and stored in cans at room 

 temperature retained its calcifying power for at least 6 months." 



Concentration. — Zucker (1922) concentrated the antirachitic potency 

 1,000 times by extracting cod-liver oil with 95 per cent alcohol, saponify- 

 ing, precipitating the calcium soaps from an aqueous solution, and 

 finally extracting the vitamin-rich material from these soaps with 

 acetone. 



Nelson and Steenbock (1925a) found that the antirachitic sub- 

 stance of cod-liver oil is non-precipitable by digitonin. 



By extracting 1,000 grams cod-liver oil with acetic or formic acid 

 and subsequently saponifying the extracted material, Dubin and Funk 

 (1923, 1924) succeeded in securing 0.5 gram of crude concentrate in 

 the form of a brown sirupy mass which on standing crystallized in 

 yellowish brown needle-like crystals radiating from a central point. By 

 eliminating the cholesterol and other inert substances the material was 

 further purified until it represented a concentration of 1 part from 

 10,000. The presence of carbon, hydrogen and oxygen was demon- 

 strated, but nitrogen, phosphorus, sulfur and the halogens were reported 

 absent. 



Koch, Cahan and Gustavson (1926) secured both from irradiated 

 cholesterol and from cod-liver oil by extraction with liquid ammonia, a 

 brown resinous substance which had great antirachitic potency. Hess, 

 Weinstock and Sherman (1926) likewise found that the ammonia- 

 soluble fraction of irradiated cholesterol was active antirachitically. 



Hess and Weinstock (1925a) reported that, "By means of spectral 

 absorption tests a chemical change was demonstrated in cholesterol 

 which had been endowed with antirachitic potency by ultraviolet irradia- 

 tion. This activated cholesterol absorbs ultraviolet radiations to a less 

 degree than does ordinary cholesterol, an effect which is intensified with 

 increasing degrees of irradiation. H, however, irradiation is prolonged 

 for many hours the activated cholesterol becomes less transparent than 

 even non-irradiated cholesterol. This comparatively opaque product is 

 of a yellowish color and has a lowered melting point." "Neither dihydro- 

 cholesterol nor dihydrophytosterol (incapable of activation) undergoes 

 a spectral change as a result of irradiation. When activated cholesterol 

 is kept in a watery suspension or in a dry state its spectral transmission 

 becomes gradually diminished until it reaches a point where it transmits 

 less than it did originally." 



In 1925 also, Steenbock and Black pointed out that excessive irradia- 

 tion (10 to 17 hours) of olive oil as well as cod-liver oil resulted in 

 their inactivation. See also Rosenheim and Webster (1926). 



