60 THE BIOCHEMISTRY OF B VITAMINS 



In view of these facts and because of the existence in nature of other 

 reacting substances, it seems unlikely that any simple colorimetric pro- 

 cedure can be worked out for the determination of all forms of vitamin B 6 . 

 Spectrophotometry analysis of the colors produced will probably have to 

 be employed. By the use of borate which under proper conditions forms 

 an inactive complex with pyridoxine only, it is possible, by difference, to 

 obtain a color value due to pyridoxine alone. 129 This has been applied 

 using the quinone chloroimide reagent. In the absence of borate under 

 prescribed conditions both pyridoxal and pyridoxamine are about one-half 

 as chromogenic as pyridoxine. 



Microbiological Methods. Pyridoxine was first shown to be a growth 

 substance for microorganisms by Moller, 130 who found it to be stimulatory 

 toward lactic acid bacteria and a bottom yeast from sauerkraut. About 

 this time Schultz, Atkin and Frey 131 and Eakin and Williams 132 inde- 

 pendently and simultaneously announced its stimulative effect on bakers' 

 yeast. A yeast growth method {Saccharomyces carlsbergensis) for its 

 determination as worked out in the Fleischmann Laboratories 133 is at 

 present the best method for the simultaneous determination of all three 

 forms of the vitamin. A yeast growth method (Saccharomyces cerevisiae) 

 was earlier developed and applied by Williams and co-workers, 134, 135 

 which contributed because of contrast with results obtained with Strepto- 

 coccus lactis (faecalis) to the discovery of "pseudo pyridoxine," later 

 identified as pyridoxal and pyridoxamine. 122, 136 



According to Snell and Rannefeld 137 and Melnick and co-workers, 129 

 pyridoxal, pyridoxamine and pyridoxine are approximately equivalent 

 in their effects on Saccharomyces carlsbergensis; but for Saccharomyces 

 cerevisiae the newer members of the group are materially less active under 

 the prescribed conditions. 



The effects of pyridoxal, pyridoxamine and pyridoxine on seventeen 

 test organisms which had been suggested or used, were studied thoroughly 

 by Snell and Rannefeld, 137 and the use of Strep, faecalis as a test organism 

 for the assay of pyridoxal and pyridoxamine was developed by Rabino- 

 witz and Snell. 114 Pyridoxal has been determined by Rabinowitz et al. 131& 

 It seems feasible by a combination of colorimetric and biological 

 methods to develop means of determining each of the three members of 

 the group. Rabinowitz and Snell have determined each of the three com- 

 ponents by microbiological means. 137b The yeast growth method {S. 

 carlsbergensis) 133 is at present reasonably satisfactory for determining all 

 three forms together, provided extraction is adequate (p. 36). 



Stokes 138 has recommended the 5-day mold test using the Neurospora 

 mutant discovered by Beadle and co-workers. For some studies the 5-day 

 incubation period would be a serious disadvantage. 



