Synthetic DL-α-lipoic acid was shown to replace the acetate to some extent in stimulating growth of Streptococcus lactis, and its acetate equivalency was determined. Although DL-α-lipoic acid itself has a strong activity, the coexistence of a small amount of thiamine promoted its activity. Even the combined action of DL-α-lipoic acid and thiamine did not however cover the activity of the sodium acetate, suggesting the probable participation of some other factor.

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Oxythiaminetriphosphate has been synthesized by treating oxythiamine with metaphosphoric acid and the competitive inhibition of thiaminetriphosphate action by this synthetic oxythiaminetriphosphate has been demonstrated in carboxylase system.

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When O, S-diacylthiamine (II) was reacted with a compound having an active thiol group, the S-acyl group of (II) was easily transacylated to acylate the latter. From these results it may be assumed that, as a part of biochemical actions of thiamine, S-acetylcocarboxylase acetylates coenzyme A by transacetylation to form acetylcoenzyme A.

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A fluorometric determination for PIN was described and the effects of various conditions were reported. Under the conditions of the determination, PIN is completely recovered through the entire procedure, PAM, PAL and lactone are practically without effect. PIC itself can interfere remarkably but in the preliminary extraction procedure it is completely converted into its lactone, thus becoming ineffective. For extraction and hydrolysis of tissue PIN, the preliminary extraction at pH 4.5 at 80° for 15 minutes and the successive hydrolysis of the filtered extract in 0.6N H₂SO₄ at 130° for 1 hour is most suitable in cases of both plant and animal tissues. For neutralizing the acid extract, the partial neutralization by barium hydroxide for removing sulfate ions is beneficial for promoting recovery.

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The method for fluorometric determination of PAM in tissues was described and the effects of various conditions were reported. The optimal condition for extracting tissue PAM is the same as described in the case of PIN. Under the condition of PAM determination, PAL is without effect.

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The fractional determination of PAL and PIC was described and the effects of various conditions were reported. PIC is adsorbed on anionic exchanger and the non-adsorbed PAL is adsorbed on cationic exchanger. PIC eluted is determined as its lactone. PAL eluted is oxidized by ammoniacal silver to PIC which is determined as its lactone. For extracting PAL from tissues, direct heating of the homogenate in 0.1N H₂SO₄ at 130° for 1 hour was found to be most suitable.
The values of 3 components of vitamin B₆ in animal and plant tissues obtained by our method were reported and compared the total B₆ values with those reported in the literature. They agreed fairly well, indicating the satisfactory specificity of the methods.

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In the urine PIN, PAL and PAM are relatively low and PIC exists predominantly. In such cases, the preliminary removal of PIC is necessary for determining each component of vitamin B₆. An appropriate method for fractional determination of 4 components are described and the conditions for it are discussed. The results of determining 4 components in the blood and urine are reported.

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The procedures for releasing and extracting vitamin B₁₂ from proteins in whole blood was investigated by the microbiological assay using Lactobacillus leichmannii ATCC 4797.
The treatment of the whole blood by heating with potassium cyanide was found to give constant values at each level of the vitamin, and good recoveries of the added vitamin in the assay.
The individual values of the vitamin in whole blood estimated by heating with potassium cyanide are in agreement with those obtained by using sodium metabisulfite and those by papain digestion, but not with those by digestion with the trypsin preparation available in this country.
The vitamin B₁₂ content of healthy human whole blood was found to range from 0.10 to 0.66mγ/ml, the average value being 0.28±0.05 (U.S.A.) and 0.37±0.07mγ/ml (Japan).

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Synthesis of dihydrothiamine (II), mp 150°, from 2-methyl-4-amino-5-aminomethyl-pyrlmldine (IV), γ-aceto-γ-mercapto-propyl alcohol (V) and formaldehyde was described. Treatment of dihydrothiamine with sodium hydroxide yielded isodlhydrothlamine, mp 160°, and both dihydrothiamine and isodihydrothiamine, when dissolved in water, were converted into pseudodihydrothiamine, mp 175°.
The three isomers, dihydrothiamine, iso- and pseudodihydrothiamine, possessed the same composition, same molecular weight, same R_F-values and reacted similarly.
They were equally converted into thiamine by treatment with ferric chloride, sodium nitraprussideor potassium ferricyanide. The action of hydrochloric acid on dihydrothiamine or its isomer was also reported.

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1. The change in vitamin B₁₂ was studied in vitamin B₁₂-deficient chicks, and the remarkable decrease of vitamin B₁₂ was confirmed in the liver, while it was scarcely observed in the blood.
2. The in vivo formation of the conjugated forms of thiamine, pantothenic acid and nicotinic acid was repeatedly tested using vitamin B₁₂-deficient chicks. Vitamin B₁₂-deficient chicks revealed a low content of conjugated vitamins in the liver, but no evidence was ever seen in the chicks treated with vitamin B₁₂. In the blood, a positive change was observed in nicotinic acid, pantothenic acid and thiamine.
3. From these findings, vitamin B₁₂ seems to be of special importance for the formation of coenzymes containing various vitamins. Further enzymic investigation is now in progress.

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