KARL FOLKERS 



carried out with micro quantities of the substance. For example, 

 early evidence concerning the non-/3-alanine portion of pantothenic 

 acid was obtained from the results of a series of new micro procedures 

 (34). Some of these new micro procedures involved determination 

 of active hydrogen atoms with deuterium oxide and of hydroxyl groups 

 with hydriodic acid, selective oxidation with iodic acid, oxidation 

 equivalent analysis, determination of a- and /3-hydroxy acids, and 

 estimation of microorganisms in suspension. The use of such tech- 

 niques represented an unconventional but fruitful approach to the 

 study of pantothenic acid and often required only one or two milli- 

 grams of the compound for each determination. 



Another type of study has been employed to open a program 

 of structural research upon a costly vitamin available in only very 

 limited quantities. This study, involving a series of "inactivation" 

 experiments, requires only a milligram or less of the substance and 

 yields information on functional groups and constitution. These 

 experiments involve adding to the micro sample of the vitamin the 

 chemical reagent (s) required for a given chemical reaction, such as 

 nitrosation or hydrolysis, and following with a microbiological assay 

 to test whether chemical reaction took place as judged by a change 

 or lack of change of activity. These "inactivation" experiments yield 

 valuable results, but they must nevertheless be interpreted with con- 

 siderable caution. They do aid in guiding the exploratory efTorts in 

 direct chemical studies. An example of such inactivation experiments 

 may be found in certain biotin studies. Brown and du Vigneaud (2) 

 described the effect of certain reagents on the activity of biotin. They 

 obtained this preliminary information with experiments on 1- or 2-cc. 

 aliquots of solution containing only 12.5 7 of biotin per cc, and the 

 criterion of reaction was the effect upon yeast growth activity. Such 

 reagents as 5% hydrogen peroxide solution, aqueous bromine, hydro- 

 chloric acid, potassium hydroxide, formaldehyde, and nitrous acid 

 caused "inactivation," indicating that a change in the structure had 

 undoubtedly been brought about by the reagent. On the other hand, 

 such reagents as acetic anhydride-sodium hydroxide, ketene, benzoyl 

 chloride-pyridine, sodium ethoxide-methyl iodide, and ninhydrin 

 caused no "inactivation," indicating that a change in structure had 

 not been brought about by these reagents. The results of these and 

 related experiments indicated to Brown and du Vigneaud that biotin 



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