BIOTIN 



it can be applied.^' * Turbidimetric methods have also been described 

 using B. radicocola ^ and Rhizohium tri/olii ^ as test organisms. 



Biotin can be estimated more satisfactorily, however, by means of 

 Lactobacillus helveticus ^ or L. arahinosus,'' as the response with these 

 organisms is measured by titrating the lactic acid produced. Improve- 

 ments in the L. helveticus method were subsequently described,® 

 including a method which was capable of assaying six members of the 

 vitamin B complex with the same basal medium.^ The L. arabinosus 

 method, although it gives lower titres than the L. helveticus method, 

 is preferred by some workers. ^° As in other assays with Lactobacilli, 

 it is essential to remove fatty material from the medium and test 

 solution, as lipoids have a stimidatory effect on the organisms. ^^ 



Other organisms that have been tested for use in the assay of 

 biotin are " cholineless " Neurospora crassa ^^ and Candida guiller- 

 mondia.'^^ Oleic acid and " Tween 80 " alone or in combination with 

 aspartic acid gave some response with the first of these organisms in 

 the absence of biotin. ^^^ 



Some of these organisms respond not only to biotin but also to 

 certain related compounds (see pages 446-454). 



The agar plate method devised by N. G. Heatley ^^ for the assay of 

 antibiotics has been used by T. I. Williams ^^ for the estimation of 

 biotin by means of S. cerevisiae. A slight modification of the method, 

 in which paper discs were used in place of cups, gave good results 

 with both 5. cerevisiae and L. arabinosus.^^^ 



In the earliest assays, extraction with hot water was used to 

 prepare the test solutions,^^ but it was subsequently found that 

 larger amounts of biotin were obtained after autolysis ^ or hydrolysis 

 or a combination of both. According to Thompson et al}'^ drastic 

 hydrolysis with 6N-sulphuric acid gives the best results, although it 

 undoubtedly causes some destruction of biotin. 



Other Methods 



No chemical or physical method of estimating biotin exists at the 

 present time. 



References to Section 7 



1. F. Kogl and B. Tonnis, Z. physiol. Chem., 1936, 242, 43. 



2. E. E. Snell, R. E. Eakin and R. J. Williams, /. Amer. Chem. Soc, 



1940. 62, 175. 



3. R. Hertz, Proc. Soc. Exp. Biol. Med., 1943, 52, 15. 



4. N. Nielsen and V. Hartelius, Biochem. Z., 1941-42, 311, 317. 



5. P. M. West and P. W. Wilson, Enzymologia, 1940, 8, 152. 



6. G. M. Shull, B. L. Hutchings and W. H. Peterson, /. Biol. Chem., 



1942, 142, 913- 



422 



