1. Two g of thiamine was decomposed by crude thiaminase of clam viscera. The several pyrimidine derivatives formed were separated by gradient elution using Amberlite XE-64.
2. Three kinds of pyrimidine derivatives were isolated in a crystalline form. One was proved to be indentical with 2-methyl-4-amino-5-hydroxymethylpyri-midine hydrochloride. Both of the other two pyrimidine derivatives gave the R_F values lower than that of thiamine, had an absorption maxima at 237 and 279mμ at pH 6.5, changed its absorption spectrum with the change of pH of the medium.

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1. A synergistic action of vitamin E for insulin with respect to glucose uptake and glycogen synthesis has been demonstrated in diaphragm from protein-depleted rats.
2. Vitamin E added as a dietary supplement brings about increases in pyruvic acid accumulated during incubation of the diaphragm, but no synergistic action for insulin in this respect was found.
3. The findings are discussed, and it is suggested that the action of vitamin E invo ves the synthesis of ATP.

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A method is described for the synthesis of four geometric isomers of vitamin A, i.e., all-trans, neo, 6-cis and 2, 6-di-cis from cis- and trans-β-ionylideneacetaldehydes and senecioate (β, β-dimethylacrylate) via intermediate vitamin A acids. The condensation of β-ionylideneacetaldehydes and senecioates was effected by means of alkali amide.

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The condensation of a variety of aldehydes with methyl or ethyl senecioate by means of alkali amide to yield γ-alkylidene- or arylidene-β-methylcrotonic acids R-CH=CH-C(CH₃)=CH-COOH is described. The ultraviolet absorption spectra of these and chemical behavior showed the arylidene compounds to have a cis relationship of the R and -C(CH₃)=CH-COOH groups about the newly created double bond, when sodium amide was used as a condensing agent. If potassium amide was a condensing agent, the results were some-what complex. In the case when R was C₆H₅, 2, 4-di-trans-acid was obtained. When R was C₆H₅-CH=CH-, the same result was obtained as the case of sodamide, namely the product was 2-trans-4-cis-acid. When R was furyl, the 2-cis-4-trans-acid was the product.
When R was alkyl and the condensing agent was potassium amide, the products seemed to be 2, 4-di-trans-acids as iodine and light failed to isomerize them.

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For the purification of the bacterial thiaminase II produced by Bacillus aneurinolyticus Kimura et Aoyama, the 7-day culture in the synthetic medium was subjected to osmotic concentration using the equipment devised by the authors and fractional saturation with ammonium sulfate. The crude enzyme obtained with 0.3-0.65 ammonium sulfate saturation was passed through each of a column of cation or anion exchange resin or through a column of both kinds of resin. By these procedures the enzyme activity was highly increased. The addition of chelate reagents, particularly EDTA, was proved to be accerelative and a crystalline substance presumably of the thiaminase could be obtained. In the process of purification the enzyme activity and absorption spectrum of each fraction were determined. Some properties of the purified preparation were reported.

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At least five fluorescent spots other than riboflavin substances were separated on the paper chromatogram of the culture filtrate of Clostridium acetobutylicum. Two of them had violet fluorescence and the remainders had green fluorescence (G, G₁ and G₂). The production of these compounds in the culture of this bacterium was parallel with the formation of riboflavin. One of the green fluorescent substances (G) promoted the synthesis of riboflavin by resting cells of this microorganism, while the violet fluorescent substance lacked such action.

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A green fluorescent substance which was found in the culture filtrate of Clostridium acetobutylicum was found to be identical with “G Compound” extracted from the mycelium of Eremothecium ashbyii and was identified as 8-N-ribityl-6, 7-dimethyllumazine. This substance was observed to have a stimulative effect on the synthesis of riboflavin by growing and resting cells of the acetone-butanol fermentation bacterium. It was also found that this substance was converted enzymatically to riboflavin by the cell-free extract of Escherichia coli when incubated with a 4-C-donor. Of the compounds tested, pyruvate was most effective as a 4-C-donor of 8-N-ribityl-6, 7-dimethyllumazine in the riboflavin biosynthesis by both microorganisms.

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First, mention was made that G Compound (2, 4-dioxo-6, 7-dimethyl-8-ribityl-pteridine) and V Compound (2, 4-dioxo-6-methyl-7-hydroxy-8-ribityl-pteridine), isolated from the culture product of Er. ashbyii, were renamed 6, 7-dimethyl-ribolumazine and 6-methyl-7-hydroxy-ribolumazine, respectively.
6, 7-Dimethyl-ribolumazine was assumed to be an intermediate in the biosynthesis of riboflavin on the basis of the results of chemical studies by Masuda and biochemical studies by Katagiri, et al. It has been recognized by many researchers that purine compounds play an important role in the biosynthesis of riboflavin. It has also been assumed that when a purine compound is added prior to the cultivation of Er. ashbyii, its C-8 is not utilized for the biosynthesis and that the pyrimidine nucleus enters as such in the isoalloxazine nucleus. So the authors investigated the relationship between 6, 7-dimethyl-ribolumazine and purine compounds, especially uracil derivatives which are the degradation products of purines.
Formation of 6, 7-dimethyl-ribolumazine is readily understood if it is considered that 4, 5-diaminouracil, a compound lacking C-8 of xanthine, is first produced, and a ribityl group then enters N-4 of the product to give 4-ribitylamino-5-aminouracil, which in turn reacts with diacetyl or with acetoin. Therefore, if such a uracil derivative is isolated from the culture products of Er. ashbyii, it is most powerful to prove the route of the biosynthesis, but in reality the formation of such a compound has not been confirmed.
According to the authors' studies, the addition of 4, 5-diaminouracil prior to the cultivation of Er. ashbyii rather checked the formation of riboflavin and neither 6, 7-dimethyl-lumazine nor lumichrome could be detected. When 4-methyl-amino-5-aminouracil instead of 4, 5-diaminouracil was added, 6, 7, 8-trimethyl-lumazine was found in the broth. In spite of the fact that this lumazine derivative readily reacts with diacetyl in vitro to give lumiflavin, no lumiflavin could be detected in the above broth. Hence, it should be considered that when a uracil derivative is converted to riboflavin via a lumazine compound, it is utilized for the biosynthesis only when its NH₂ at C-4 is substituted by a ribityl group.

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1. The vitamin B₁₂ content of the erythrocytes from 20 normal men has been found to vary from 135 to 230μμg per ml of the packed erythrocytes with a mean value of 195μμg.
2. Vitamin B₁₂ in human erythrocytes exists in the hemoglobin fraction in some combined form with protein which has an electrophoretic mobility similar to that of hemoglobin.
3. Heat stability in pH 6.8 phosphate buffer of the combined vitamin B₁₂ in the hemoglobin fraction has been investigated. Release by heat of the combined vitamin B₁₂ takes place more readily in pH 4.6 acetate buffer than in pH 6.8 phosphate buffer, although the release by 30 minutes heating in both buffers is almost equal.
4. The solution of the crystallized human oxyhemoglobin has a limited capacity to bind the added vitamin in vitro. The added vitamin which is bound in the hemoglobin solution can be recovered from the hemoglobin band on paper strips by a paper electrophoretic technique.
5. From the results it is suggested that vitamin B₁₂ in human erythrocytes exists in a combined form with hemoglobin.

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1. Using crude melanin separated from human hair, vitamin A was proved to be adsorbed fairly well on the melanin. The adsorbed vitamin could be liberated from melanin with ethyl ether without suffering oxidation or destruction.
2. The same experiments were also carried out with the liver melanin isolated from the black-colored liver of the dogfish as well as with active carbon, and similar results were obtained.
3. The adsorbability of vitamin A on active carbon, hair melanin, casein, liver protein and other substances in petroleum ether or in aqueous solution (solubilized by Tween 80) was compaired. The following order of adsorbability was obtained in both solvents:
Active carbon>hair melanin, liver melanin>casein, liver protein, gelatin>powdered agar-agar, glass powder, defatted cotton wool.
4. The order of adsorbability of natural vitamin A liver oil containing glyceride on melanin or other substances in petroleum ether was fairly the same as in the case of the vitamin alone, though the adsorbability was somewhat decreased.

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