190 VITAMIN D GROUP 



specific only as to classes. Reactions of interest in the study of ergosterol 

 and/or other provitamins D include the following : the Tanret or reversed 

 Salkowski reactionj^"** • ^'^^ the Liebermann-Burchard reaction/""- ^^e. 262 ^j^g 

 Tortelli-Jaffe reaction,^^^ • 266, 267, 268 ^j^g Rosenheim reactions (trichloroacetic 

 acid or chloral hydrate) ,2*^ • ^^^ the Rosenheim-Callow reaction,-^^' 2™ the 

 Tschugajeff reaction/^- ^^i and the antimony trichloride reaction.-'*^ ■ 2^^- ^^^ 

 A modification of the trichloroacetic acid reaction, notable for its extreme 

 sensitivity, is described by Christiani and Anger .^^^ Most of these reactions 

 distinguish the provitamins D from sterols of the cholesterol-phytosterol 

 class. Some distinguish provitamins from their esters,^^^' ^'^^ and oxidized 

 from fresh specimens of provitamin. ^^^^ ^^^^ ^^^ 



For the quantitative estimation of ergosterol, three general methods are 

 available: (1) color reactions, (2) digitonide precipitation, and (3) spectro- 

 photometric measurement. No method is known w^hich will distinguish 

 minute amounts of ergosterol from other provitamins D, but when sufficient 

 material is on hand, the esters and oxidation products can sometimes be 

 identified. The color reactions have the advantage of being applicable to 

 minute quantities, but they are subject to considerable error. Heiduschka 

 and Lindner^"" used the Liebermann-Burchard reaction for the determina- 

 tion of ergosterol in yeast, a questionable procedure because this reaction 

 is given not only by ergosterol but (more slowly) by sterols with a single 

 double bond.2^^ Bilger et al}'^^ used the Liebermann-Burchard reaction for 

 the total sterols of yeast, and a modification of the Rosenheim trichloro- 

 acetic acid reaction for the ergosterol. Page^''* applied a modification of the 

 Rosenheim reaction to the estimation of ''ergosterol" in animal tissues; see, 

 however, the comments of Bilger. The more recent (1939) Christiani- 

 Anger^^^ modification, in which the chloroform solution of the unknown is 

 treated with trichloroacetic acid and lead tetraacetate, deserves a quanti- 

 tative development. In spot tests it detects as little as 0.1 7 of ergosterol, 

 and it distinguishes esters from free sterols. 



Ergosterol, like all sterols of normal steric configuration, gives with 

 digitonin a precipitate of low solubility which lends itself to gravimetric 

 estimation. The digitonin reaction is accurate but time-consuming. It does 

 not take place with esters, and thus when applied to tissue extracts before 



«6 E. P. Haussler and E. Brauchli, Helv. Chim. Acta 12, 187 (1929). 



267 I. M. Heilbron and F. S. Spring, Biochem. J. 24, 133 (1930). 



2«8U. Westphal, Ber. 72, 1243 (1939). 



269 0. Rosenheim, Biochem. J. 23, 47 (1929). 



"0 O. Rosenheim and R. K. Callow, Biochem. J. 25, 74 (1931). 



"1 L. Tschugajeff, Chem. Z. 24, 542 (1900); Z. angew. Chem. 13, 618 (1900). 



2'2 A. F. V. Christiani and V. Anger, Ber. 72, 1124, 1482 (1939). 



2" F. Bilger, W. Halden, and M. K. Zacherl, Mikrochemie 15, 119 (1934). 



274 I. H. Page, Biochem. Z. 220, 420 (1930). 



