A general method for the synthesis of pyridoxal adenosine diphosphate and related compounds is described. It consists of the reaction of adenosine-5-phosphoromorpholidate with pyridoxal-5-phosphate, pyridoxamine-5-phosphate and pyridoxine-5-phosphate in anhydrous pyridine or dimethylformamide. The method has been applied to the preparation of pyridoxal-, pyridoxamine- and pyridoxine adenosine diphosphate. The yields of nucleotides were generally satisfactory, varying between 40 and 52%.

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The reaction of inosine-5-phosphoromorpholidate with pyridoxal-5-phosphate in anhydrous pyridine gave pyridoxal inosine diphosphate in 35% yield, after 15 hours at 40-50℃. Similarly, it was synthesized from pyridoxal adenosine diphosphate by deaminization with nitrous acid in 40% yield. Irradiation of alkaline solution of pyridoxal inosine diphosphate under aerobic condition yielded mainly pyridoxic acid inosine dinucleotide. The latter was obtained by the reaction of inosine-5-phosphoromorpholidate with 4-pyridoxic acid-5-phosphate in 28% yield.

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Additive tests of mononucleosides on the growth rate of various strains were first carried out for L. leichmannii assay of vitamin B_<12>, using thymidine, inosine, adenosine or guanosine. Positive effect was seen by an addition of thymidine, but was more effective by additions of both vitamin B_<12> and thymidine. Additive tests of both vitamin B_<12> and various ribonucleosides to Skeggs' modified medium were found to accelerate the growth rate of the strain in 3-4 hours less than a single addition of vitamin B_<12>, while effects were scarcely observed by a single addition of the ribonucleosides. In L. arabinosus assay of pantothenic acid, additive effects of the ribonucleosides were scarcely recognized, on the contrary, they rather acted inhibitory on the growth. In Stc. faecalis R assay of folic acid, positive influence of thymidine on the growth of the strain was always demonstrated, and more effective by addition of both folic acid and thymidine. Concerning the addition of both vitamin and ribonucleoside, detectable influence was always found. In Sacch. carlsbergensis assay of vitamin B_6,marked response of ribonucleosides on the strain was also observed.

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In previous papers, the present authors reported that the relationship between vitamin D potency and sulfate metabolism was studied and higher level of sulfate in keel and left tibia of chicks receiving vitamin D_3 was demonstrated using ^<35>S-sulfate. Afterwards, further researches were reported for the action of vitamin D_ in active sulfate metabolism of chicks. The present paper deals with further studies on the microdetermination of vitamin D by paper chromatography. Successful results were obtained for the separation and estimation of vitamin D, provitamin and other sterols using paper chromatography.

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In this study, the present authors first carried out oral experiments of vitamin D_3 with rabbits and the urinary volume and calcium output were estimated. The excretion of vitamin D_3 in the animals following the administration of vitamin D_3 in quantity in excess of the compound normally ingested was studied. The urinary vitamin D_3 was not demonstrated in lipid-soluble form but the vitamin was found in water-soluble conjugated form. In parallel experiments urinary sulfate and uronate levels were tested. The findings have 1ed to assume that there may be urinary excretion of vitamin D_3-conjugates in animals following the administration of vitamin D_3

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The addition of L-ascorbic acid (10^<-2>-10^<-3>M) to the reaction medium was found to promote greatly the white mustard myrosinase activity under such a condition (pH 6.4) as to produce neither white turbidity nor hydrogen sulfide. The amount of the white turbidity and hydrogen sulfide produced on addition of the ascorbic acid to the reaction mixture of myrosinase and sinigrin increased with the decreasing pH value of the medium. Either of those materials was produced in a larger amount in the case of white mustard enzyme than in the case of mold enzyme. The material producing white turbidity was found to be composed mainly of protein. It may be concluded that the ascorbic acid is a specific activator of myrosinase.

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S-Methylmethionine sulfonium chloride (MMSC) itself had no methionine activity on L. arabinosus, but showed the activity only when homocysteine was added. This function was charactiristic of MMSC and homocysteine, and choline, betaine or carnitine did not replace MMSC, nor did thioglycolate and cysteine. From these results, the microbioassay of DL-MMSC ranging 10 to 50μg was established. L. arabinosus was used as test microorganism and 200μg/tube of DL-homocysteine was added. The stabilities of MMSC to acid, alkali and heat were observed. MMSC was stable at room temperature. It was, however, labile at 100℃ except in acidic solution. The determination of MMSC in commercial preparations was tried.

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Column chromatography with Amberlite IRA-411 (OH^--form) was used to remove contaminations such as methionine or methionine sulfoxide which were active to the microbioassay of S-methylmethionine sulfonium chloride and the recovery of the latter through this treatment was excellent. Chemical assay of the latter compound which was based on ninhydrin color reaction was able to use for the determination of commercial preparations except those containing diastase, by using chromatographic treatment. Data between microbioassay and chemical assay were about the same.

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Growth of Lactobacillus arabinosus, Lactobacillus casei, Streptococcus faecalis, and Leuconostoc mesenteroides were investigated using alanine-free synthetic media containing essential vitamins. These four lactobacilli required vitamin B_6 for their growths. So the growth promoting effects of pyridoxal, pyridoxamine and pyridoxine were compared. In the absence of vitamin B_6,alanines had varying effects on the growth. Any form of alanines had no effect for L. casei and a little effect for L. arabinosus. DL-Alanine promoted the growth of Stc. faecalis sufficiently but either D- or L-alanine remained ineffective. For Leuc. mesenteroides alanines were moderately effective. On the other hand, D-alanine exhibited inhibitory effect on the growth of L. casei in a semisynthetic medium. From these experimental results, it can be said that for the studies of anti-vitamin B_6 activities of various compounds, the alanine-free synthetic media are more preferable to the semisynthetic media.

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In the preceding paper the production of DBCC was reported by cultivation of Propionibacterium shermanii in a medium containing hydroxocobalamin or cyanocobalamin as a precursor. This paper deals with the production of DBCC by cell suspension of the bacterium. Under the exeprimental condition almost complete conversion of hydroxocobalamin to the coenzyme was achieved in a concentration as high as 50μg/ml, which was about 10 times higher than that of the growing cell method. As the concentration of the precursor increased moreover, the degree of the transformation decreased and the formation of an unidentified vitamin B_<12> analogue was observed. The effectiveness of cyanocobalamin as the precursor was only one third of that of hydroxocobalamin in this method.

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When TATD is incubated with washed blood cells, a detectable amount of free thiamine has been noted in the surrounding fluid in addition to intracellular accumulation of thiamine. Mechanism of the appearance of the extracellular thiamine was investigated. Results showed that the factor(s) which was formed from the reaction of TATD with the blood cells contributed to the reduction of residual TATD in surrounding medium. The factor(s) appears to be the reduced form of the lipoate moiety (or its catabolite) liberated from TATD by the cells. Reduction of TATD and iso-TATD by washed blood cells was inhibited by SH-blocking agents. No considerable effect is observed by using KCN and 2,4-dinitrophenol. Whereas arsenic acid did not affect the TATD reduction by glutathione, the thiamine liberation from TATD by blood cells was depressed effectively by this reagent. This suggests the concerning of vicinal dithiol as well as monothiol in the TATD reduction by the blood cells.

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TATD was reduced easily by excess amount of glutathione to liberate thiamine quantitatively. The reaction was accelerated according to the elevation of pH from 3.8 to 7.5. On the reaction with excess of TATD the molar amount of liberated thiamine was equivalent to that of added glutathione. Treatment of TATD with glutathione in equimolar concentration, in neutral or weakly acidic medium, resulted in the formation of a new ninhydrin-positive compound (X) possessing biological activity corresponding to that of lipoic acid. When TATD was replaced by lipoic acid in similar condition the formation of such a product has not been observed. From the results of qualitative investigation X substance appears to be mixed disulfide between glutathione and lipoate moiety of TATD.

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The level of lipoic acid activity in blood after the intravenous injection of TATD was higher and long lasting than after the injection of lipoic acid. This high activity in blood was predominantly distributed in the plasma fraction and a considerable part of its activity was found to be combined with plasma protein. Namely, when the plasma after 30 minutes of the injection of TATD was treated with ethanol or 5% metaphosphoric acid to precipitate the protein, about 30% of the activity in the plasma was found in this protein fraction. Thus, the combination of the lipoate moiety with the plasma protein should be responsible for the long maintenance of the high activity in blood after the injection of TATD.

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A titration method for the determination of lipoic acid using Streptococcus faecalis R was investigated. The medium used for Streptococcus faecalis 10C1 was also applied which showed a better sensitivity for growth of Stc. faecalis R than that of Stc. faecalis 10C1. A suitable standard response curve after 24 hour incubation was ranged from 0.025 to 0.5mμg/ml. Growth stimulating activity of some lipoic acid derivatives was also examined. Assay for lipoic acid in natural materials gave a satisfactory result in urine, however, those in blood and liver were varried to some extent depending on the extraction methods and dilution of samples.

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