II. CHEMISTRY 139 



amounts. He found that cholesterol purified via the dibromide, or via allo- 

 cholesterol, and sitosterol purified via the dibromide, did not show the usual 

 absorption. 



These studies made it plain that an impurity present in ordinary choles- 

 terol and sitosterol is responsible for at least the greater part of the anti- 

 ricketic activity conferred upon the sterols by irradiation. The impurity, 

 or provitamin, was presently identified as ergosterol, a sterol which 

 Rosenheim and Webster-^ had previously found to be activatable, but with 

 which they had made no quantitative studies. PohP'' found that the three 

 known absorption bands of ordinary cholesterol are also exhibited by ergos- 

 terol, but in vastly greater intensity. Additional chemical studies and a 

 biological assay were made by Windaus and Hess,^^ who found that, as 

 expected, the antiricketic potency of irradiated ergosterol was far greater 

 than that of irradiated cholesterol. Rosenheim and Webster^'- also conducted 

 chemical and biological studies and concluded that the precursor of vitamin 

 D "is ergosterol, or a highly unsaturated sterol of similar constitution." 



Bills et alP confirmed these studies in a way which supported the view 

 that ergosterol was the contaminant provitamin. By the use of a continuous 

 light source they discovered that ordinary cholesterol exhibits a fourth 

 absorption band, X 260 m/x. The same band was shown by ergosterol, making 

 the series XX 293.5, 282, 270, and 260 m;u, with four points of identity in- 

 stead of three. Moreover, the spectra faded at the same rate when acetone 

 solutions of ordinary cholesterol and of provitamin-free cholesterol plus 

 added ergosterol were oxidized with permanganate. 



In spite of all the e\'idence, it turned out that the identification of the 

 provitamin of cholesterol as ergosterol was incorrect, yet it was so con- 

 vincing that it was generally accepted from 1927 to 1934. However, there 

 was during that period a growing knowledge of the multiple nature of 

 vitamin D, which has been reviewed elsewhere in some detail.^* In par- 

 ticular, Massengale and Nussmeier^* observed that activated ergosterol is 

 much less effective than cod liver oil, rat unit for rat unit, in the prevention 

 of rickets in chicks. They offered no explanation of this phenomenon, but 

 their work provided an important tool, the chick-rat efficacy ratio, for 

 future studies on the D vitamins. 



It remained for WaddelP® to demonstrate that the provitamin D of 



" O. Rosenheim and T. A. Webster, Biochem. J. 20, 537 (1926). 



30 R. Pohl, Nachr. Ges. Wiss. Gottingen, Math, physik. Kl. 185 (1927). 



'1 A. Windaus and A. Hess, Nachr. Ges. Wiss. Gottingen, Math, physik. Kl. 175 (1927). 



'2 0. Rosenheim and T. A. Webster, Lancet I, 306 (1927); Biochem. J. 21, 389 (1927). 



" C. E. Bills, E. M. Honeywell, and W. A. MacNair, J. Biol. Chem. 76, 251 (1928). 



" C. E. Bills, Cold Spring Harbor Symposia Quant. Biol. 3, 328 (1935). 



" O. N. Massengale and M. Nussmeier, /. Biol. Chem. 87, 423 (1930). 



" J. Waddell, /. Biol. Chem. 105, 711 (1934). 



