Uptake of thiamine-^3H hydrochloride (B_1-^3H-HCl) and O-benzoylthiamine disulfide-^3H (BTDS-^3H) in both central and peripheral nerve tissues of the mice with oral administration has been studied by microautoradiogram. In the brain and spinal cord two pathways of the vitamin were observed, namely through the capillaries and liquor. In the cerebrum uptake of the vitamin in cortex and basal ganglia was more marked than in white matter. Nerve cells and glia cells exhibited the distinct uptake, though it was more marked in the former. Uptake of the nerve cells was different in degree, according to their histological localization or individual saturation state of the cells. In the cerebellum uptake of B_1-^3H and BTDS-^3H was most marked in Purkinje cells and then in granular layer. In spinal cord nerve cells of each column exhibited marked uptake. In lower neuron, uptake of the vitamin was also distinct in perikaryon, which sent the vitamin into the axon of nervous fibers by axon flow. Thiamine in myelin sheath was supplied by Schwann cells. The motor end-plate revealed more marked uptake than the sensory ending did. In peripheral autonomic neuron the uptake of the vitamin was markedly demonstrated in the perikaryon. In central and peripheral nerve tissues the uptake of BTDS-^3H was more markedly observed than that of B_1-^3H.

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Simplification of Fujita's method in the bacterial thiaminase test was attempted. Removement of bacterial cells from culture fluids may be abbreviated. Conditions of the reaction were unified in 2/75M phosphate buffer at pH 7.0,40℃ for 60 minutes. However, the optimum conditions should be adopted in the case of assay for each thiaminase activity. Pyridine (10^<-4>M, final concentration) was added in the reaction system in order to enhance the activity of the thiaminase I. It showed neither the inhibition for the activity of thiaminase II nor the disturbance for the estimation of thiochrome. Diastase digestion for the check of phosphorylation of thiamine can be omitted. The simplified method or the direct method in the previous paper was employed for the assay of remaining thiamine after the reaction. Thus, it was demonstrated that the simplified method of bacterial thiaminase test was employed as the substituted method for the Fujita's method because of its convenience and equal sensitivity.

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Pyrithiamine (I), when incubated with the culture medium of Kloeckera apiculata, was accumulated on cells 1.7 times as much as thiamine. The accumulated amounts of anti-thiamine compounds other than (I) were less than thiamine ; bezylimidazole-thiamine (II) : 0.67,butylimidazole-thiamine (II) : 0.45,thiamine sulfuric ester (IV) : O.09,triazino-thiamine (V) : 0.07,phenyltriazino-thiamine (VI) : 0.06,imidazole-thiamine (VII) : 0.04,desthiothiamine (VIII) : 0.02,oxythiamine (IX) : 0.02,in comparison with thiamine. When Kloeckera cells preincubated with (I) or (II), were washed and transfered to a thiamine solution, the cells released (I) or (II) gradually, as thiamine was accumulated increasingly on the cells. These anti-thiamine compounds except (IX), inhibited cell-uptake of thimine, when they were incubated with the culture of Kloeckera and thiamine. Inhibition ratios of the compounds were shown in percentage ; (II) : 87,(I) : 67,(VII) : 62,(III) : 58,(IV) : 24,(VIII) : 13,(VI) : 11,(V) : 7. From these results, it was concluded that anti-thiamine compounds of the stronger growth inhibition index to Kloeckera were more strongly accumulated on the cells and inhibited cell-uptake of thiamine more strongly at the presence of thiamine.

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Some of modified thiamine compounds stimulated the growth of Kloeckera apiculata more strongly than thiamine but the growth by these compounds was more effectively inhibited by oxythiamine than the growth by thiamine, inhibited by oxythiamine. 5O% inhibition index to oxythiamine ; thiamine, 68,000 ; thiamine propyldisulfide (TPD), 4,600 ; benzoylthiamine monophosphate (BTMP), 210 and S-carbethoxythiamine (CET), 42. The accumulation of these compounds on Kloeckera cells was inhibited by oxythiamine ; inversely proportional to inhibition index. Kloeckera cells, when preincubated and accumulated with TPD or BTMP, were inhibited by oxythiamine on the growth test to the same extent as thiamine, whereas cells preincubated with CET were inhibited more strongly by oxythiamine than those with TPD or BTMP. These differences could be explained by the stability of CET and easy reversion of TPD or BTMP to thiamine.

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An additional study was done on the microbiological determination using Kloeckera apiculata which was described by Hoff-Jφrgensen and was modified by us (Vitamins 23,213,1961). Amount of thiamine in inoculum culture medium was changed to 0.2μg/2ml, instead of 1.0μg/2ml in previous report. An addition of vitamin-free yeast extract to the basal medium increased the cell growth. Thiamine-free yeast extract was prepared as follows. 50ml of 10% yeast extract is autoclaved at 120℃, 3Omin. after an addition of 0.25g of sodium sulfite, followed by removal of excess sulfite by hydrogen peroxide. Slant culture(about 10°) increased the sensitivity about two times as high as usual culture. The both cultures were useful for determination of 10-5Omμg of thiamine while shaking culture for 1-10mμg.

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The antirachitic potency of human milk was tested by the prophylactic and curative methods using white rats which were previously fed on vitamin D-deficient diet. The animals were orally given with 2ml of human milk (containing 780 I.U. of vitamin D per litter). The results were always realized to be positive by X-ray test and A.O.A.C. ash test.

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Based on the results of the previous report, the mixture of amino acids in place of casein hydrolysate in Atkin's basal medium that afford equal growth response to pyridoxine, pyridoxal, pyridoxamine for each other was studied. When histidine and tryptophan were eliminated from the previous reported basal medium, growth response to the each form of vitamin B_6 was equal together. Vitamin B_6 contents that were determined by using the medium containing glutamic acid, aspartic acid, asparagine, arginine, methionine, isoleucine, and ammonium sulfate as nitrogen source agreed closely with those of the casamino acid medium.

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Under the various concentrations of thiamine, pyridoxine, and pantothenic acid in the vitamin B_6 assay medium, the growth, contents of vitamins in the cells of Saccharomyces carlsbergensis 4228 and the alcoholic fermentation were examined during the incubation time. At the presence of thiamine in the medium, the growth degree of the yeast was varied according to the concentration of pantothenic acid, and alcoholic fermentation was stimulated. The content of thiamine in the cells reached the highest at the lag time and then decreased with the progress of the incubation. Vitamin B_6 biosynthetic ability of the yeast was the most active at the absence of both thiamine and pyridoxine in the medium.

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During the L-malic acid production in CO_2-fixing fermentation by Schizophyllum commune, a blue coloration of mycelium was seldom seen, and at the same time malate formation was reduced. The earlier the pigment was formed, the more marked decrease of malate production was observed. Addition of lipoic acid, pantothenic acid and thiamine in the medium caused the stimulation of sugar expense and the inhibition of malate formation, as previously reported, but the blue coloration caused the lowering the former and strengthening the latter. Inhibitory effect of Cu^<2+> on malate formation was not affected by blue pigmentation. An addition of ethanolic extract of blue mycelium caused a marked decrease of malate production. From these results, it was supposed that the pigment may promote the reverse reaction of phosphoenolpyruvate carboxylation. The pigment resembled to indigo derivative. When mucilage was produced in the medium, an increase of malate production was observed.

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When retinoic acid in chloroform solution was reacted with sulfuric acid, a red color (λ_<max> of 540 mμ) was produced in the sulfuric acid layer ; E^<1%>_<1cm>(540 mμ) 1240. The color was stable for 20 minutes but upon the addition of water, the color produced by retinoic acid faded away rapidly. This color reaction of retinoic acid was more reproducible than SbCl_3-reaction. Recovery tests of retinoic acid by using rat liver homogenates were proved to be satisfactory for routine determination.

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When retinoic acid in chloroform solution was reacted with different concentration of sulfuric acid (75-95%), the sulfuric acid layer showed two maximal peaks of absorption at 440mμ and 540mμ, respectively. The absorbance at 440mμ increased and the peak was shifted to 450mμ with dilution of sulfuric acid. When 74% sulfuric acid was used, the maximal absorbance at 450mμ was obtained, while the peak at 540mμ disappeared. The yellow color of the 74% sulfuric acid layer was stable and intensity of the color was completely proportional to the concentration of retinoic acid. From the results, it was concluded that retinoic acid could be determined quantitatively by colorimetric assay of 74% sulfuric acid layer at 450mμ.

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The deleterious effect of pyridoxine-deficiency on antibody formation has been demonstrated by many authors in mice, rats and guinea pigs, while the mode of action of the vitamin has not been elucidated. The author studied about immunological phenomena in pyridoxine-deficient rats and monkeys and tried to clarify the mechanism of inhibitory effect of the deficiency on antibody formation, in relation to thymus function. In pyridoxine-deficient animals, thymus atrophy, small lymphocytes depletion of peripheral blood and immunological unresponsiveness are observed which means a remarkable decrease of immunologically competent cells. In pyridoxine-deficiency, the thymus atrophy is prominent and specific and it may cause the impairement of antibody formation. The author propose to name this phenomena as "nutritional thymectomy".

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Activities of glutamic-oxalacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) in the supernatant and mitochondrial fractions of the liver and kidney of vitamin B_6-deficient rats were investigated. Young male albino rats were used as the material. In a group of the rats, a vitamin B_6-deficient diet was administered for 5-6 weeks. In another group of the rats, an injection of 20mg of penicillamine which is the antagonist of vitamin B_6,was given once a day and repeated on successive two days. In both groups of the rats, the activities of transaminases were markedly decreased in the soluble fraction of liver and kidney cells, but in contrast with soluble fraction not definitely decreased in the mitochondrial fraction. Further, changes in transaminase activities by the addition of pyridoxal phosphate to the cell fractions of B_6 deficient rats were examined. Approximately 20% increase of the activity was observed in the soluble fraction of the liver, but in the mitochondrial fractions no significant elevations were observed.

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It was found that thiamine 8-(methyl 6-acetyldihydrothioctate) disulfide (TATD) reducing activity in erythrocyte hemolysate was heat stable and above 90% of it was inhibited by N-ethylmaleimide and p-chloromercuribenzoate. Experimental results from dialysis treatment of the hemolysate showed that reduced glutathione (GSH), out of sulfhydryl compounds in erythrocytes, held a main part of TATD reducing activity. When erythrocyte was incubated with TATD, consumption of GSH in erythrocytes has been noted according to the increase of added TATD concentration. This change of GSH level was also observed by using of thiamine propyldisulfide in place of TATD. The presence of glucose or inosine suppressed apparently the consumption of GSH. Restoration of GSH level was observed by incubation of erythrocytes in Krebs-Ringer phosphate buffer (pH7.4) after the repeated washings of the erythrocytes separated from reaction mixture with TATD and the restoration was accelerated by the addition of glucose or inosine in incubation medium. No remarkable differences of total glutathione (reduced plus oxized) were found among the intact-, TATD treated- and restored-erythrocytes. This suggests that the consumption of GSH by the incubation of erythrocytes withTATD reflects the change of GSH to its oxidized form and the recovery of reduced form is proceeded by glutathione reductase system in erythrocytes which is probably stimulated through the enhanced supplying of NADPH by glucose or inosine.

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Thiamine liberation from thiamine 8-(methyl 6-acetyldihydrothioctate) disulfide (TATD) in erythrocyte hemolysate, which contained glucose-6-phosphate dehydrogenase coupled glutathione reductase (GSSG-reductase) system, was accelerated by addition of NADPH or NADP plus glucose-6-phosphate. On the other hand this acceleration effect was not observed in dialyzed hemolysate. However the acceleration effect was reappeared by addition of limited amount of reduced glutathione (GSH) to dialyzed hemolysate. Acceleration effect by NADPH under the presence of catalytic amount of GSH was also observed on thiamine liberation from thiamine propyldisulfide and thiamine disulfide in dialyzed hemolysate. Activities of GSSG-reductase system were found in dialyzed and non-dialyzed hemolysate in reasonable amount for these acceleration effects. Addition of TATD stimulated ^<14>CO_2 production from glucose-1-^<14>C and -2-^<14>C by wased erythrocytes suspension. These results show a occurrence of the coupling mechanism between GSH oxidation by thiamine disulfide derivatives and glucose-6-phosphate dehydrogenase coupled GSSG reductase system in erythrocytes.

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