Determination of Vitamin B, by Yeast Fermentation Method Improvements Related to Use of Sulfite Cleavage and a New Fermentometer ALFRED S. SCHULTZ, LAWRENCE ATKIN, AND CHARLES S . FREY The Fleischmann Laboratories, Standard Brands Incorporated, New York, N. Y.
The fermentation activity of thiamin as measured by the method of Schultz, Atkin, and Frey is approximately 99 per cent destroyed by treatment with sulfite at pH 5 to 6 and at 100"C. for 30 minutes. The known interfering substances are not affected by this treatment. Thiamin assaSs are based on the meas-
T
HE yeast fermentation met'liod for the determination of thiamin is based on the observation (2) that the rate of
urement of fermentation activity before and after sulfite treatment. This technique has been applied to a wide variety of materials, the thiamin content of which may be of interest. A small compact ferrrientometer has been designed, to make the yeast fermentation method available for routine use.
equivalent to that portion of the fermentation activity of the snhstnnce which can be destroyed by treatment with sulfite.
alcoholic fermentation by living yeast may be increased by the addition of t.hiamin chloride, and is one of tlie first microbiological assay methods to be successfully employed. Like most other biological methods, it is highly specific under suitable conditions. The only interfering substrmce of any significance which has thus far been found (3) is 2-methyl-5etlioxymethgl-6-aminopyrimidirie. The corresponding 5Iiydroxymethgl compound is a hydrolysis product of thiamin and is also active in the fermentation reaction. Except for itl; possible existence in urine, this type of substance is not found to any great extent in nature. The int'erference caused by it may be overcome by oxidation of t'lie vitamin B, with alkaline ferricyanide (6, 5 ) or treatment with sulfite (6). Both procedures depend on the chemical inactivation of thiamin by tlie re:igents mentioned and det'erniination of the interfering substance in tlie residue. The sulfite procedure is preferred because it is more convenient. nnd is applicable to a wider variety uf material?. The sulfite cleawge O C C U ~ Rin the follon-in# m:inner ( 7 ) :
Method of Assay
Two preparations are made of each substance to be analyzedone treated with sulfite and one untreated-and then fermentation activities are det'ermined with the aid of a fermentometer. T!re difference, expressed in terms of pure thiamin chloride, is taken as a measure of the thiamin content of the unknown. P R E P A R A T IOF~ S SAMPLES. A water-soluble substance is dissolved in a convenient volume of water (depending upon the potency of the sample). Insoluble materials must be finely divided either in the dry state by grinding or in the wet by means of the Karing Hlendor or its equivalent. The latter procedurebas heen found particularly useful in the preparation of bread and tissue samples. The suspension should be dispersed well enough to permit the measurement of an accurate aliquot by means of a pipet. The solution or suspension is made acid to Congo red paper with sulfuric acid and heated a t 100" C . for 20 minutes. Tbis is conveniently done in an Arnold steam sterilizer. When cool the solution is made to volume and a portion is taken for sulfite treatment (described below). The remainder is neutralized to neutral litmus paper by means of sodium hydroxide and is then used for the determination of total fermentation activityi. e., before sulfite treatment. TREAmiEwr WITH SULFITE.I n a 50-ml. Pyrex Erlenmeyer C"3 flask is placed an aliquot (not exceeding 20 ml. in volume) of the I I sample prepared as above, and 0.7 ml. of N sulX=C--NII,.HCl c==c.:-cH~(~H~O1[ furic acid and 5 nil. of a freshly prepared sohI f /' tion of sodium sulfite containing 0.2 gram of CH,C -c ( ' 1 1 2 -1t Xa.rSOa , ~\ Sa2S0,.7H,O per 5 ml. are added. The reaction is adjusted to pH 5.2 t o 5.6 with dilute sul"["--S sI' --CHI! furic acid or sodium hydroxide solutions, using C'1 bromocresol purple as an outside indicator to set the pII. The flask is capped with a 30-ml. beaker, Thi:unin Clhloride heated at 100" C. for 30 minutes, and then cooled. Excess sulfite is destroyed by the addition of the exact quantity of a 3 per cent CH, solution of hydrogen peroxide. The end point of the reaction is I determined with the aid of an outside indicator made by mixing X==C-NH, C=C--C'H,
