ASSAY METHODS 63 



biotin" in "Vitab," rat brain, beer or Squibb urease, and warned against 

 the use of high dosage levels in applying the yeast test. As larger and 

 larger amounts of extracts are introduced into the yeast medium, the test 

 becomes less specific, because of the existence of other yeast nutrilites 

 which may not be in any way related to biotin. 155 



The existence and significance of the "uncombinable biotin" in urine 

 has never been clarified. Probably the most reasonable explanation at the 

 present time is that in urine there exist biotin degradation products which 

 do not combine with avidin and yet can be utilized by yeast in lieu of 

 biotin. The existence of such substances has never been completely clari- 

 fied and their interference with other asasy methods has not been ade- 

 quately studied. 



Hofmann and Winnick 156 have utilized the fact that oxybiotin is active 

 for yeast in the determination of oxybiotin in the presence of biotin. 

 Biotin is destroyed by dilute permanganate under conditions that oxybio- 

 tin is unaffected, so that the response after oxidation is a measure of the 

 oxybiotin present. 



2. Methods Utilizing Lactic Acid Bacteria. Several papers have been 

 published describing biotin assay methods or modifications thereof, which 

 utilize L. casei as the test organism. 157 - 158 > 159 > 16 ° Difficulties are encoun- 

 tered in obtaining a biotin-free basal medium, and the same interferences, 

 particularly of fatty substances, occur as with other tests involving this 

 organism. If the titration method is used for evaluating responses, more 

 time is required to obtain results than in the yeast test. 



More recently Wright and Skeggs 161 have developed the use of L. 

 arabinosus for biotin assay. Because the basal medium needs to contain 

 only known substances, and for other reasons, this organism appears to 

 have distinct advantages. A recent survey (1945) is reported to indicate 

 that seven out of nine prominent laboratories use L. arabinosus for biotin 

 assays. 152 However, the amount of published analytical data accumulated 

 using this method is small, and any shortcomings that the method may 

 possess can be discovered only by extensive use. Because of the nature of 

 the medium and high sensitivity of the organism to biotin, one would not 

 anticipate any serious difficulties in the general application of the method. 

 In the test as originally described, 161 titration after 72 hours was used to 

 evaluate responses. Subsequently, Wright 152 has indicated that satisfac- 

 tory results may be obtained turbidimetrically after about 24 hours of 

 growth, or by titration after 48 or even 24 hours. Speeding up the 

 response in this test could doubtless be accomplished by suitable modifica- 

 tions (if necessary) of the improved medium for L. casei developed by 

 Roberts and Snell. 50 



Other biotin assays have utilized as test organisms Rhizobia, 154 Clos- 



