1. Separation of urine and feces from poultry droppings was attained by fitting an artificial anus with cannula, which prevented the excretion of ordinary droppings, thus it has become possible to determine the net content of urinary thiamine in hens.
2. Addition test of thiamine to the urine showed the recovery to be 97 to 98%.
3. After oral administration of thiamine, the urinary excretion of the vitamin was 15% of the intake. However, the administration of nonthiazolium type analogues, i.e., thiamine propyl disulfide, benzoylthiamine monophosphate and dibenzoylthiamine was found more effective.

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Young male rats of Wister strain were kept at 20° as a control group and at 0 to 5° as test groups. They were beheaded after 1, 6, 24 hours and 21 days. Relation between the unusual metabolism of adrenal cortex hormones affected by low temperature and the effects of vitamin A supplement was studied.
1. The ascorbic acid content in the adrenals increased from one hour exposure to cold, showing the maximum after six hours, and fell to a normal level gradually thereafter. It showed a greater decrease in the case of a vitamin A-deficient dose and rose again with vitamin A supplement.
2. On the metabolism of adrenal cortex hormones, the experimental defects of Rabinovici's report were pointed out and the influence of stress was examined quantitatively, including endocrine accumulation in the adrenals and urinary excretion.
3. The volumes of the urine were increased by cold, and diuretic action was found to be affected by vitamin A.
4. As to the influence of cold-stress, the remarkable changes in the contents of corticosteroids and ascorbic acid in the adrenals occurred during 1 to 24 hours. Thereafter, the test animals seemed to become accustomed to the environmental changes.

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The enzymes obtained from a cell-free extract of bakers' yeast catalyzed the condensation of the pyrimidine (OMP) and thiazole (Th) moieties of thiamine to form thiamine. ATP and Mg²⁺ were required essentially for the reaction and Mn²⁺ also showed a fair activity. An optimal pH of the reaction was 7.0. Some other pyrimidine compounds which differ from OMP in the chemical structure at the C₅-position of pyrimidine are less active than OMP as the substrates. The enzymatic activations of OMP and Th by ATP in a preliminary step before the condensation were demonstrated.

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1. Phosphates of 2-methyl-4-amino-5-hydroxymethylpyrimidine (OMP) and 4-methyl-5-β-hydroxymethyl thiazole (Th) were synthesized chemically and the activities of thiamine synthesis without ATP were examined. OMP pyrophosphate (OMP-PP) and Th phosphate (Th-P) were most active as substrates and had a reaction rate of 5 times as much as that of the thiamine synthesis from OMP and Th with ATP. OMP-PP and Th pyrophosphate (Th-PP) had also a fair activity as substrates.
2. The thiamine synthesized from OMP-PP and Th-P was proved to be a mixture of thiamine and thiamine monophosphate, while that from OMP-PP and Th-PP a mixture of thiamine, thiamine monophosphate and thiamine pyrophosphate.
3. The active compound of OMP was isolated by Dowex 1 column chromatography from the enzyme reaction mixture incubated with OMP-P³² or ATP-γ-P³². The isolated OMP-PP formed from OMP-P³² and ATP, and OMP-P and OMP-PP formed from OMP and ATP-γ-P³² were identified by using paper chromatography, bioautography with a E. coli mutant, actigraphy and absorption spectra.
4. Comparing the specific radioactivities of the OMP-P and OMP-PP isolated with those of OMP-P³² or ATP-γ-P³² added, OMP-PP is presumed to be mainly formed by successive phosphorylation of the γ-phosphate of ATP through OMP-P.
5. The active intermediate of Th enzymatically formed was identified bioautographically as Th-P and the mechanism of enzymatic synthesis of thiamine was discussed.

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1. Purification of the thiaminase I produced by B. thiaminolyticus Matsukawa et Misawa has been described. By applying the 70% ammonium sulfate fraction of crude thiaminase I to chromatographic fractionation using an ion exchange resin, Duolite A2, and zone electrophoresis, an enzyme preparation having 200 times as much specific activity as that of the original crude enzyme was obtained. Ultracentrifugal analysis indicated that the preparation contained a single component and was free from any organic bases. The purified thiaminase alone does not show any thiaminase activity, but its activity appears in the presence of an organic base (pyridine) leading to the formation of heteropyrithiamine.
2. K_m for thiamine and pyridine of the purified enzyme were 0.9×10^-3 and 1.0×10^-3M at pH 6.5 and 30°, respectively, according to Lineweaver and Burk's method. V_max was 190mg/min/mg protein-N, expressed as the amount of thiamine decomposed.
3. The sedimentation constant and other observations suggest a molecular weight of approximately 40, 000.
4. According to the results of inhibition experiments, the enzyme is considered to be an SH-enzyme.

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1. Observation was made to find whether the pyridoxine palmitates had the atoxoprimidine (ATXP) activity in mice.
2. It was found that the sensitivity of mice to 2-methyl-4-amino-5-hydroxymethyl pyrimidine (OMP) increased by feeding a synthetic vitamin B₆-deficient diet for 7 days was depressed by feeding the deficient diet supplemented with the pyridoxine palmitates equimolecular to PIN-HCl.
3. In the experiments of oral and subcutaneous administrations in a single dose at various intervals before and after the OMP injection, it was found that the PIN palmitates exhibited the ATXP effects when administered one to 24 hours before the OMP injection and that the maximum effect was seen 6 hours before the OMP injection, while the activity of pyridoxine hydrochloride was maximum when administered simultaneously with OMP and exhibited no activity when administered 24 hours before OMP injection.
4. In the above experiments, the ATXP activity of pyridoxine dipalmitate was stronger than pyridoxine tripalmitate.
5. When the pyridoxine palmitates was injected intraperitoneally in a single dose, only a slight activity of ATXP was observed.
6. It was suggested that the apparent ATXP activity of the PIN palmitates was possibly due to the pyridoxine produced by hydrolysis in vivo.

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1. It was found that riboflavin was biosynthesized by various green leaves from 6, 7-dimethyl-8-ribityllumazine without participation of other carbon sources.
2. It was established that the synthetic reaction, observed with a crude preparation from spinach leaves, was catalyzed by an enzyme. It has an optimum pH of 7.5 and follows the first order kinetics.
3. The stoichiometry of the vitamin synthesis from the substrate was determined and a hypothetical scheme of the mechanism for the biogenesis was proposed.
4. The effects of a numbr of substances on the enzyme reaction were studied.

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Test dose experiments were conducted on both undernourished and well-nourished boys. The mean thiamine or riboflavin excretion after the test dose was found significantly higher in undernourished boys than in the well-nourished. Administration of cocarboxylase or flavin-adenine dinucleotide, however, diminished the difference between both groups.
The authors ascribe the increased excretion of thiamine or riboflavin in undernourished boys after dosing to the decreased ability of phosphorylation in their bodies.

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Pyrimidinyl-cysteine was detected by paper chromatography when thiamine was decomposed in the presence of cysteine by partially purified thiaminase I prepared from Bacillus thiaminolyticus.
The reaction product from thiamine and cysteine by purified thiaminase I was isolated from the reaction mixture in crystalline form, mp 219-220° (decomp.), by means of column chromatography using Amberlite IRC-50. The crystalline substance was found hardly soluble in organic solvents and cold water, but soluble in hot water, and in an acid or alkaline solution. The substance gave positive reaction with both Dragendorff and ninhydrin reagents, but negative with nitroprusside even after treatment with zinc powder in acid solution. Its R_F value was shown to be 0.36 by ascending paper chromatography with a n-butanol-methanol-water system (25°, 16hr) when an acid solution of the crystal was spotted.
The isolated compound was found to be identical with an authentic sample of pyrimidinyl-cysteine in melting point, elementary analyses, and absorption spectra, etc. The chemical constitution of pyrimidinyl-cysteine was shown to be S-(2-methyl-4-amino-pyrimidinyl-(5)-methyl)-cysteine. S-(2-methyl-4-amino-pyrimidinyl-(5)-methyl)-cysteine sulfoxide, formula II, was obtained as an oxidation product of pyrimidinyl-cystine. The infrared absorption spectrum of the sulfoxide showed the existence of a sulfoxide group in the molecule.
It was clearly shown that thiaminase I catalysed an exchange reaction between the thiazole moiety of thiamine and cysteine, probably also other sulfhydryl compounds, besides the well-known base exchange reaction.

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A simplified method is proposed for the rapid determination of vitamin A in animal tissues in the presence of a large amount of β-carotene, using a modified method for dehydration of vitamin A. Special care for the exclusion of moisture was not required, since the dehydration reagent used was alcoholic hydrochloric acid containing a small amount of water. Evaporation process was not needed, because the dehydration reaction was carried out in a benzene solution and the sample solution was not diluted with the reagent. Single extraction with benzene was enough to extract vitamin A and β-carotene almost completely from the digested homogenate. The recoveries of vitamin A and β-carotene added to the tissue homogenates prior to the alkali digestion were satisfactory for ordinary biochemical researches. Application of this method to the semimicro determination of vitamin A is also presented. The determination of the amounts of vitamin A and carotene in about 10 samples were possible in about 6 hours.

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