In medium containing D- and L- alanine (or DL-alanine) the growth curve of Leuconostoc mesenteroides exhibits an unusual phenomenon in the presence of the vitamin B₆ antagonist, 2-methyl-4-amino-5-hydroxymethylpyrimidine (OMPm). Increasing the concentration of the inhibitor, it increasingly inhibits growth up to a certain level, after which growth of the organisms resumes and increases until the amount of inhibitor is approximately doubled; with further increase of the amount of the inhibitor above this level, growth of the bacteria is again restricted.
The same characteristic curve was obtained with other antagonists of pyridoxal, 2-methyl-4-methylamino-5-hydroxymethylpyrimidine and isonicotinic acid hydrazide.
The appearance of the characteristic phenomenon was not related to the presence of vitamin B₆. It occurred when the vitamin was supplied as pyridoxal, pyridoxal phosphate, pyridoxamine or pyridoxine, as well as when it was omitted completely from the medium.
This phenomenon was not observed in cultures of Streptococcus faecalis R.

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1. In the presence of glyoxalic acid and copper ion, pyridoxamine phosphate (PAM-P) was promptly converted into pyridoxal phosphate (PAL-P) non-enzymatically with high yield.
2. In this reaction, formation of yellowish brown PAL-P-glyoxalic acid-Cu-chelate from PAL-P, glyoxalic acid, and copper ion was suggested by the ultraviolet absorption spectra and the analytical data.
3. Pyridoxal phosphate-isonicotinic acid hydrazone (PAL-P-INAH) was obtained by the reaction of isonicotinic acid hydrazide after PAM-P was oxidized by colloidal MnO₂. After removing the protecting group by 0.1N HCl and silver nitrite, PAL-P was purified by column chromatography using Dowex 50W-X8 (H⁺-form). This method, however, gave lower yield.
4. Pyridoxamine, isopropylidene-pyridoxine, or deoxypyridoxine was allowed to react with P₂O₅+85% H₃PO₄. Without separating the polyphosphate ester, the reaction mixture was hydrolyzed by 30% phosphoric acid which was produced by addition of water to the reaction mixture. Monophosphate ester thus formed was purified by baryta, charcoal, and Amberlite column chromatopraphy and PAL-P, PIN-P, and deoxypyridoxine phosphate were obtained with high yields.

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Growth of Lactobacillus arabinosus, Lactobacillus casei, Streptococcus faecalis, and Leuconostoc mesentèroides were investigated in alanine-free synthetic media containing essential vitamins. These four lactobacilli required vitamin B₆ for their growth. So the growth-promoting effects of pyridoxal, pyridoxamine, and pyridoxine were compared. In the absence of vitamin B₆, alanine had varying effects on the growth. Any form of alanine had no effect on L. casei and a little on L. arabinosus. DL-Alanine promoted the growth of S. faecalis sufficiently but either D- or L-alanine remained ineffective. On Leuc. mesenteroides, however, each form of alanine was moderately effective. On the other hand, D-alanine exhibited inhibitory effect on the growth of L. casei in the semisynthetic medium. From these experimental results, it can be said that for the study of anti-vitamin B6 activities of various compounds, alanine-free synthetic medium is preferable to the semisynthetic one.

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1. By the injection of 5mg of riboflavin in the femoral muscle of a rabbit, flavin quantity in blood increased very rapidly, and 30% of the injected amount of riboflavin was excreted into urine in one hour and 60% of that in 6 hours.
2. In the case of riboflavin-tetrapalmitate injection, almost no increase of flavin in blood and urine was observed.
3. In the case of riboflavin-tetrabutyrate injection, an appreciable increase of flavin in blood and urine was observed. Within 24 hours after the injection, 30-40% of the injected amount was excreted into urine as free riboflavin.
4. From these facts, it is possible to employ riboflavin tetrabutyrate as a riboflavin derivative possessing the desired depositing action in the body.

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