V. ESTIMATION 509 



deiivatives.-'^ The (3-, y-, and 6-nitrosotocopherol.s can l)e separated by 

 simple chromatography, estimated separatelj^ and the difference between 

 their sum and the total as determined l^y the Emmerie and Engel method 

 is a-tocopherol."'^ Recently^^ eountercurrent distribution has been employed 

 for the separation of tocopherol from tocopherylquinone in stools. The 

 a, 0, and 8 compounds have been cleanly separated by reversed-phase 

 paper chromatography, but the /8 and y isomers could not be."*^ 



Fluorometry has also been applied, preferably to the unsaponifiable mat- 

 ter of fats after chromatographic separation ; the nitric acid oxidation prod- 

 uct is treated with o-phenylenediamine to form the fluorescent phenazine.^^ 



If the purely physical methods for estimation of the tocopherols are limi- 

 ted to their absorption spectra in the ultraviolet region, the first and ap- 

 parently only attempt to apply the technique ciuantitatively to natural 

 products^^ showed that unidentified substances seriously interfere both 

 before and after saponification. 



2. Bio ASS AY 



Tocopherol, the "fertility" vitamin, owes its name to the first demonstra- 

 tion of vitamin E deficiency, the failure of laboratory rats to procreate. 

 This was a chance observation, in Pasteur's sense, and the existence and 

 indispensability of vitamin E might have been discovered in connection 

 with several other unnatural and diseased conditions now known to be 

 produced when it is lacking. Indeed, deprivation of vitamin E is followed 

 by a more baffling array of physiological abnormalities in different species 

 than has ever been encountered with a single vitamin. None of these dis- 

 orders is as suitable for purposes of bioassay as the restoration of fertility 

 in a vitamin E-deficient female rat. In one or another of its modifications, 

 this circumstantial, tedious, and demanding procedure has been the guide 

 and milestone in the exploratory work on the distribution and chemical 

 nature of vitamin E. 



To be valid, such a bioassay must meet several criteria.*" 

 1. Fertilization by a normal male must be established by the finding of 

 sperm in the \'aginal contents or of a vaginal plug. 



** J. V. Scudi and R. P. Buhs, /. Biol. Chetn. 146, 1 (1942). 



" M. L. Quaife, /. Biol. Chem. 175, 605 (1948). 



" H. Rosenkrantz, A. T. Millhorat, and M. Farber, /. Biol. Chem. 192, 9 (1951). 



" F. Brown and K. L. Blaxter, Chemistry & Industry 1951, 633. 



« .VI. Kofler, Helv. Chim. Acta 25, 1469 (1942); 26, 2166 (1943); 28, 26 (1945) ; 30, 1053 



(1947). 



H. Lieck and H. Willstaedt, Svensk Kern. Tidskr. 67, 134 (1945) [C.A. 40, 4759 



(1946)]. 

 *^T. Moore and K. R. Rajagopal, Biochem. J. 34, 335 (1940). 

 " A. L. Bacharach, E. Allchorne, and H. E. Gl^nn, Biochem. J. 31, 2287 (1937). 



