ESTIMATION 



Microbiological Assay of Pyridoxine in presence of Pyridoxamine 

 and Pyridoxal 



It will be clear from the foregoing that the estimation of vitamin Bg 

 activity by measuring the growth response of micro-organisms may 

 give misleading results, as different organisms respond in different 

 degrees to the three substances. The lactic acid bacteria, for example, 

 are useless for this purpose, as they show a greater response to pyri- 

 doxamine and pjnridoxal than to pyridoxine. The method of M. Landy 

 and D. M. Dicken,^^ which utilises L. helveticus, is invalid for this 

 reason. By means of a special medium L. helveticus can be used for 

 the assay of pyridoxal. ^^° 



Perhaps the most satisfactory method of estimating pyridoxine in 

 presence of its two derivatives is that of Stokes et al.,^^ in which use 

 is made of an X-ray induced mutant of Neurospora sitophila ^' that 

 requires pyridoxine but does not respond to pyridoxamine or pyri- 

 doxal. The medium is relatively simple, consisting of sucrose, am- 

 monium tartrate, ammonium citrate, inorganic salts and a minute 

 amount of biotin ; it was modified slightly by E. C. Barton-Wright.^^ 

 The response is measured by weighing the dried mycelium. The 

 results obtained were in good agreement with those obtained by 

 biological assays. An important point to be borne in mind when 

 carrying out assays with N. sitophila, is that aneurine must be de- 

 stroyed with sodiimi sulphite, as the organism does not respond 

 quantitatively to pyridoxine in the presence of aneurine ; residual 

 sulphite must be destroyed by addition of hydrogen peroxide. 



L. E. Carpenter and F. M. Strong ^^ confirmed the claim of Stokes 

 et al. that Neurospora assays gave results in good agreement with those 

 obtained by the rat growth method, but they failed to obtain con- 

 sistent results and expressed a preference for the yeast growth method. 



Pyridoxine, pyridoxal and pyridoxamine can be separated by 

 partition chromatography on a strip of filter-paper and the position 

 of the spots detected by laying the filter-paper for a few minutes on 

 the surface of an agar plate seeded with 5. carlsbergensis and then 

 incubating the plate. ^^ Growth of the organism is stimulated around 

 the areas in which the three substances are concentrated ; pyridox- 

 amine is held near the top of the paper and pyridoxine near the bottom 

 with pyridoxal just above it. 



References to Section 7 



1. C. E. Edgar, M. M. El Sadr and T. F. Macrae, Biochem. J., 1938, 



32, 2200. 



2. R. C. Bender and G. C. Supplee, /. Nutrition, 1940, 20, 109. 



3. T. W. Conger and C. A. Elvehjem, /. Biol. Chem., 1941, 138, 555. 



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