For the determination of vitamin A in pharmaceuticals, Mulder's method has the advantage of rapidity and accuracy as compared with U.S.P. method. However, it tends to give higher values in some of the preparations. The authors proved that the cause of the higher values is chiefly involved in the washing process with KOH, i.e., benzene is dissolved into the aqueous phase to some extent, and not vitamin A, thus resulting in a relative concentration of vitamin A. This phenomenon was not observed when cyclohexane was used as the extracting solvent instead of benzene. The cyclohexane method applied to U.S.P. vitamin A reference standard and other various forms of vitamin A preparations resulted in good agreement with those of U.S.P. method.

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The studies of the effect of vitamin B₁₂ administration to a large number of Japanese children in different areas were carried out from 1959 to 1961 by the B₁₂ Study Group of the Vitamin B Research Committee of Japan.
It was observed that oral administration of 25μg of B₁₂ daily to the children aged 3 to 14 years over a period of one year did not show any definite promoting effect on the growth of the children. However, in the statistical analyses for B₁₂ values of the whole blood, 276-380 children aged 9 to 14 years showed a significant increase in the B₁₂ level when B₁₂ was supplemented for 6 months to one year.
The average normal value of B₁₂ in the whole blood of Japanese children aged 9 to 14 years was found to be 0.361mμg (δ=0.135mμg)/ml.
As the children in this study were taking daily around 1μg or more of B₁₂ from their diet, the findings mentioned above do not exclude the possibility of growth-promoting effects on children when the intake of the vitamin is not sufficient.

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Applying equilibrium dialysis at pH 3 to the combination of O-benzoylthlamine disulfide (BTDS) or its derivatives with protein, the results in agreement with the previous findings were obtained. The results of showed the combination of protein with BTDS or its derivative to be mostly due to the SS-linkage.

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A vitamin B₁₂-like substance, tentatively called X factor, was isolated from an animal feed supplementing B₁₂. From the results of paper electrophoresis, absorption spectra, microbiological activities, precursor effect in B₁₂ biosynthesis. X factor was shown to have a structure nearly related to cobinamide. Though a number of B₁₂-like substances have so far been isolated, such as from sewage and culture medium of Propionibacterium shermannii (Bernhauer) and from supernatant liquor distiller's waste digester and human excreta treatment plant (Shimizu), but the isolation of such large quantities of cobinamide has not been reported. Especially, the method adopted by the authors using P-cellulose is very simple and the separation is satisfactory. It can be safely recommended for preparation of cobinamide.
TABLE III Effect of X Factor as a Precursor in Cobammide Biosynthesis
It is not clear whether this cobinamide has been present in the feed supplement or has released from cobamide during purification procedures. If the former is the case, the significance as an intermediate in cobamide biosynthesis must be considered. Studies on the mechanism of cobamide biosynthesis using X factor and the promotion of cobamide production by culture are the problems under way. As paper electrophoresis of X factor in a neutral medium resulted often in separation of several spots and that in an acidic medium whithout cyanide resulted in appearance of yellow spot, X factor may not be a simple compound.

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X factor isolated from a bacterial cell preparation is cobinamide, or a closely related compound. Its addition to the culture medium together with dimethylbenzimidazole resulted in a marked increase in cobamide production by P. Shermanii, a cobamide-producing organism. The highest value obtained under optimum condition was as high as 5 μg/ml, a yield not very high in view of industrial production, but considering the fact that the cobamide production by P. Shermanii IAM 1713 used by the authors is not very high, producing insignificant cobamide in the medium of Bernhauer devoid of cobalt salts. The pronounced increase in cobamide production after addition of X factor and DBI seems to suggest both compounds to be the precursors. One of the purpose of the authors is to identify the form of cobamide produced and the yield of the cobamide may be sufficient for such study.

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After injecting the labeled thiamine propyl disulfide, TPD-S³⁵(outer), in the ligated intestines of dogs and rats, the total and free thiamine was measured. The amount of S³⁵ was estimated in the contents of the ligated intestinal canal, the ligated portion of the intestine and the blood of mesenteric vein from the ligated portion. The results show that TPD moves rapidly into the tissues, where it is then reduced to thiamine, separating the propylmercapto radical.
Matsubara (3) submitted a hypothesis concerning the absorption of TPD that the compound might be mainly reduced to thiamine in the intestinal canal, but some of it may pass through the intestinal canal in the form of TPD. A part of the reduced TPD might be resynthetheized to TPD, and the S-alkylmercaptocysteine, produced by reduction of TPD, may aid the absorption of thiamine in the intestinal canal.
The results of our experiments revealed that a majority of the TPD administered entered the intestinal walls without being reduced, although some part of it may be reduced in the intestinal canal. The propylmercapto radical of TPD was separated shortly after its invasion into the intestinal canal. The S³⁵ of the propylmercapto radical separated from TPD was excreted rapidly after absorption. 80 to 90 per cent of the S³⁵ was excreted in urine and feces during a 41-hour period following oral administration of TPD-S³⁵ (outer). However, 50 to 80 per cent was excreted in the urine and feces within 48 hours after intramuscular injection. In the contents of the small intestine, only 0.5-0.8 per cent of the administered radioactivity was found. The radioactive S³⁵ found in the viscera following intramuscular injection were 1.5-4.5 per cent. Consequently, higher counts of S³⁵ were found in the urine and feces after oral administration than after intramuscular injection.
Marked discrepancies were noted when the results obtained after intravenous and oral administrations were compared. The findings suggest that TPD passes through the tissues in various ways according to the mode of administration. A part of the propylmercapto radical is separated when TPD was absorbed from the intestine and excreted through the lymphatic system.

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1. The rate of the synthesis of riboflavin from G compound became independent of the presence or the absence of cysteine and ascorbic acid under anaerobic condition.
2. The content of molecular oxygen in the enzyme solution was promptly decreased by the addition of cysteine or ascorbic acid, and, more remarkably, of both the reagents.
3. It was demonstrated that cysteine and ascorbic acid have no intrinsic role as the activators in the biogenesis of riboflavin, but their existence causes the reaction medium to be anaerobic, and, consequently, the substrate was almost exclusively utilized for riboflavinogenesis.

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1. It was found that V compound could be easily produced through a chemical reaction of G compound and p-quinone under physiological conditions, which might be possible to be extended to the consideration of the biosynthesis of V compound.
2. The chemical reaction was demonstrated to be a dehydrogenetic demethylation reaction by chemical kinetics studies on the reaction.
3. The most plausible mechanism for the chemical reaction was proposed.

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1. Flavokinase activity was found in various green leaves, and some properties of the crude enzyme preparation obtained from the spinach were investigated.
2. Mg ion was essential for the reaction, and both ATP and ADP were effective as the phosphate donor for this preparation, while AMP was a competitive inhibitor in the presence of ATP.

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1. Bean flavokinase has been highly purified to the specific activity of 80 mμmoles FMN formed per hour per mg protein at 30° by protamine treatment, fractionation with ammonium sulfate and chromatography on DEAE-cellulose. This preparation did not contain phosphatase activity and practically behaved as a homogeneous system in electrophoresis and ultracentrifugation.
2. The optimum pH was around 9.0 and the optimum temperature for the enzyme activity was abount 40°.
3. The metallic ions, especially Mg²⁺ was essential for full activity of the enzyme.
4. ATP was shown to be the specific phosphate donor. While ADP had a slight effect on the phosphorylation, GTP, CTP and ITP were entirely ineffective.
5. The enzyme was half saturated with riboflavin at the concentration of 1.3×10^-5 M.

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1. The effects of pyrimidine mononucleotides on liver-pyruvate oxidase system was studied, and it was found that the addition of UMP or CMP resulted in similar increase of the enzyme activity. The metabolic fate of the nucleotides in the reaction mixture was quite similar to the case of inosine-5′-phosphate.
FIG. 2 Effects of Mononucleotides on the Activation of Thiamine Diphosphate in Rat Liver-Pyruvate Oxidase System
FIG. 3 Paper Chromatograms of the Metabolic Products of Various Mononucleotides in the Reaction Mixture of Rat Liver-Pyruvate Oxidase System
Solvent I, n-butanol-acetic acid-water (12:3:5); Solvent II, water (pH 5.2 to 5.5); Solvent III, n-butanol (water-saturated); Solvent IV, pyridine-n-butanol-water (1:1:1).
Ad-R, adenosine; Ad, adenine; G-R, guanosine; G, guanine; I-R, inosine; U-R, uridine; U, uracil; C-R, cytidine; C, cytosine.
2. The effects of various nucleosides on the enzyme activity were also tested, and the degradation products were estimated by paper chromatography. The metabolic fates of the mononucleosides were found to be quite similar to the case of inosine, and the reappearance of the mononucleosides was noticed after addition of both the corresponding bases and D-ribose.

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1. Using pure standard preparations it was found that vitamin A acid could be produced from natural vitamin A aldehyde by treating with a dilute alkaline solution according to Cannizzaro's reaction, judging from the findings of spectrophotometry and paper chromatography.
2. Metabolic pathways of β-carotene, vitamin A aldehyde and vitamin A alcohol under anaerobic condition were studied using the intestine of rats, and it was demonstrated that the terminal fission of β-carotene might take place, yielding proteinbound vitamin A aldehyde and β-ionone.
3. Further, the same experiments were performed under aerobic condition, and the fates of β-carotene and vitamin A were studied. The results suggest that at the final step of vitamin A metabolism in the intestine, protein-bound vitamin A aldehyde might be converted to vitamin A alcohol and vitamin A acid.

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On the basis of dehydration reaction, the fatty acids of vitamin A esters were selectively separated from other coexisting lipid esters such as glyceride. Fatty acid composition was determined by the use of gas chromatography. Vitamin A esters in fish liver oils were composed of various saturated and unsaturated fatty acids from C₁₄ to C₂₄. Dominant members were palmitic and oleic acids. Polyenoic acid content was generally low as compared with that of other lipid esters such as glyceride.

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It was proved that riboflavin-tetrabutyrate has the same vitamin action as riboflavin, but the action of riboflavin-tetrapalmitate was not observed clearly.
The ariboflavinosis-curing effect of riboflavin-tetrabutyrate was also demonstrated, but riboflavin-tetrapalmitate did not cure the ariboflavinosis.

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