3084
NOTES
We wish t o thank Mr. E. B. Damon and Dr. W. A. Ray for the kind loan of apparatus used in this investigation. HARVARD C H E M I C a L LABORATORIES
Vol. 60
The Specificity of the Fermentation Test for Vitamin B1 BY ALFRED s. SCHULTZ,
1,AWRENCE -4TKIN AND C H A R L E S
N. FREV
CAXBRIDGE, hIASSACHUSETTS
The fermentation method for the determination of vitamin has been in successful operation for some time. The effect of 2-methyl-5-ethoxymethyl-6-aminopyrimidinehas been described.2 We have assayed a wide variety of substances Changes in the Physical Properties of Regener- such as non-autoclaved yeast, rice polish, vitamin ated Cellulose by Liquid Ammonia pills and concentrates, solutions of crystalline BY RICHARD G. ROBERTS vitamin, and milk, and no evideiice of the iiiterDuring the dialysis of proteins and hormones fering substance has been found. I n an invcstiwe contained in bags made from regenerated cellulose gation on the metabolism of vitamin B1,4,5 found reason to believe that a portion of the ferfilni and placed in liquid ammonia, it was observed that the bags changed in size and in flexibility. mentation stimulating effect of urine is iiot due t o Therefore, a series of comparative tests on some of vitamin B1. While this did not appear to alter the physical properties of the film and ammonia- the significance of the results, it was thought very treated film was made. The film used in these desirable to find a method for differentiating between the intact vitamin molecule and any postests was du Pont Cellophane number GOO. sible breakdown product. way of doing this The film was cut into strips of convenient size has been found in the differential oxidatioii of the and placed in a Dewar flask. Liquid ammonia, vitamin Bl in the presence of the aminopyrimipreviously dried over metallic sodium, was added dine. to immerse the film sample completely. The Alkaline ferricyanide in the cold will readily film was iiot previously dried by us. The Dewar oxidize the vitamin to thiochrome. Preliminary flask was tightly stoppered, and attached t o a experiments with a sample of thiochrome obtained mercury seal. The liquid ammonia boiled off in from hierck and Company showed it to be inacabout twenty-four hours, and any excess ammonia tive in the fermentation reaction. The aminopygas was removed by a vacuum pump. rimidine is more resistant to oxidation and it is a Physical properties showing an increase: simple matter to oxidize B1 preferentially when Tear streiigth (Illmendorf test). . . . . . . . . . . . . 200.07, present in addition to aminopprimidine. A soluTensile strength.. ........................ 70.6% tion containing 8 gamma of the amiiiopyrimidiiie Thickness (flat micrometer). . . . . . . . . . . . . . . . 152.4% and 8 gamma of thiamin hydrochloride in a volWeight (per unit area). .................... 27.3% ume of 35 ml. was treated with 2.5 ml. of 1% Physical properties showing a decrease: K3Fe(CN)e and 2.5 ml. of 50% NaOH. After 8.3% standing at room temperature for five minutes Length (with grain). ...................... W d t h (cross grain). ...................... 17.87, the solution was neutralized with dilute sulfuric Area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24.77, acid and made t o 100 ml. A 25-ml. aliquot of Ratios: Increase of tear strength to decrease in this mas tested in the usual manner by gas test. area, approximately 8 to 1. Increase of tensile It gave a stimulation LT-hich corresponded esactly strength to decrease in area, approximately 3 to 1. to 2 gamma of the aminopyrimidine (i. e., the B1 It has been shown that bags made from regen- was destroyed) Parallel experiments showed erated cellulose film may be used conveniently for that the neutralized oxidizing solution was withdialyzing experiments in liquid ammonia, although out influence on controls with either thiamin hydrochloride or aminopyrimidine. some space must be allowed for shrinkage. The author wishes t o thank Montgomery Ward (1) A. S. S c h u l t z , L . Atkiil a n d C. N. F r e y . THIS J O U R N A L , 69, 948 and Company, Chicago, Illinois, for the use of (1937). (2) A. S. Schultz, L. Atkin a n d C. X . F r e y , i b i d . , 69, 3457 (1937). apparatus in their testing laboratory. (3) A. S. Schultz, L. Atkin a n d C. N. F r e y , i b i d . , 6 0 , 1514 (1938). GEORGE DAVISSCIEXCE HALL Ksox COLLEGE GALESBCRG, ILLINOIS RECEIVED JULY 18, 1938
A%
THEDEPARTMENT OF CHEMISTRY MEDICAL SCHOOL CHICAGO CHICAGO, ILLISOIS RECEIVED AUGUST29, 1938
(4) A. S. Schultz, R. F. Light and C. S. Frey, Proc. SOC.E x p l l . B i d . M e d . , 38, 404-406 (1938). ( 5 ) R. F. Light, A. S. Schultz. J,. Atkin and L. J. Crams, J . Nurr., 16, 333 (1938).
