ASSAY METHODS 61 



Biological Assay. The only biological assay method that has been 

 used extensively and which has been applied to pyridoxal and pyri- 

 doxamine is the method of Conger and Elvehjem 139 which has recently 

 been improved by Sarma, et aL 139a This involves the growth response of 

 rats which had been depleted 4 to 6 weeks on a basal diet containing 

 B vitamins in the form of thiamine, riboflavin, nicotinic acid, choline and 

 pantothenic acid in pure form and 1:20 liver concentrate powder. The 

 growth response is linear with respect to the amount of vitamin B 6 added. 

 As originally designed, this test, like the microbiological tests, was for 

 the assay of pyridoxine, on the assumption that pyridoxine and vitamin 

 B 6 were identical. Pyridoxamine and pyridoxal are about equally active 

 in the test, however, so it constitutes a method for determining all three 

 forms of the vitamin simultaneously. Assay values obtained by this 

 method for very few duplicable materials are available, and a wider 

 application of the test, particularly to low-potency materials might easily 

 show it to possess serious flaws. The biological method of Dimick and 

 Schreffer, 140 which appears to be less specific, has also been employed in 

 some recent studies. 129 Elvehjem has recently suggested an improved basal 

 diet for the rat growth test. 141 This has been investigated in only a pre- 

 liminary way. 



Biotin 



The only available methods for the assay of biotin involve the use of 

 microorganisms or higher animals. The outstanding obstacle to the devel- 

 opment of a chemical method which would be applicable to natural 

 material is the fact that biotin occurs naturally in exceedingly low con- 

 centrations which are beyond the reach of most kinds of chemical tests. 



Microbiological Tests: 



1. Yeast Growth Method. Yeast was the test organism used ini- 

 tially in the discovery and isolation of biotin by Kogl and co-workers. 142 

 However, the assay method utilized fresh yeast (heavy seeding) obtained 

 directly from a brewery and hence was not applicable to laboratories in 

 general. The most widely used assay method was that developed in the 

 Texas laboratories, which utilizes a pure culture of bakers' yeast. 143 



The basal medium for this yeast test contained only known substances, 

 including inositol, /^-alanine (synthetic calcium pantothenate was not yet 

 available), thiamine and pyridoxine; 16-hour growth responses were 

 obtained from the addition to 12-ml cultures of biotin in amounts of 25 

 to 250 micromicrograms (10 ~ 12 gram). This method has the advantage of 

 speed and is also specific to a high degree, since biotin is an extremely 

 potent nutrilite and the amounts of material (in the form of a tissue 



