The protective effect of N-pyridoxylidene-L-cysteine against X-ray irradiation was recognized from the survival percentage, when N-pyridoxylidene-L-cysteine was injected intraperitoneally to mice 5 minutes before X-ray whole body irradiation.

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Observations were made on histological changes in the liver and spleen of the Vitamin B_6 deficient rats which were injected subcutaneously with 15 mg of L-cysteic acid per kg. of body weight on alternate days. Swelling of the spleen and enlargement of its follicles were recognized. These results suggest that vitamin B_6 prevents the injury of liver and spleen due to L-cysteic acid.

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The ingested vitamin A appears rapidly in serum, reaching a maximum level about 2-3 hours after an intake. The maximum level rises almost proportionally to the amount of the vitamin ingested.

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Changes in the carotene and vitamin A levels in serum after ingesting varying amounts of β-carotene together with breakfast were examined and no significant rise in the two was observed so long as the amount of β-carotenedid not exceed 2 mg. Following an ingestion of 4-8 mg β-carotene, the carotene level rose distinctly, reaching a maximum after about 5 hours, but the rise in vitamin A level was not so marked, reaching a maximum after about 3-4 hours. Further increases in the amount ingested up to 124 mg resulted in distinct, but relatively small, rises in serum carotene levels, while the rise was insignificant in serum vitamin A levels. The course of the rise in serum carotene levels after receiving 4 mg β-carotene was found to be roughly the same as that after receiving cooked strained carrot containg 4.3 mg α-carotene and 4.0 mg β-carotene, but the rise in serum vitamin A levels was somewhat less after ingesting the cooked carrot. After an intake of 0.5-4mg β-carotene, the maximum was attained in 4-6 hours in the carotene level and 3-4 hours in the vitamin A level. The absolute levels of plasma vitamin A were somewhat higher than those of carotene. The rise in plasma vitamin A levels after ingesting carotene was about 30% of that after ingesting the same I. U. of vitamin A.

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In acute experimental CCl_4 poisoning of rabbits the urinary excretion of pantothenic acid decreased. It reached the lowest level on the 4th day and returned to normal by the 7th day. In chronic liver damage, the urinary pantothenic acid decreased gradually until the end of the experiment (3 monthes later). The increase of urinary pantothenic acid following the intramuscular administration of calcium pantothenate was also depressed in both groups of animals. In acute histologically severe cases, the increase was found to be greater than that of the control animals. The pantothenic acid content of liver, especially the bound form, was less than normal in both groups. These results led to the conclusion that abnormal metabolism of pantothenic acid may exist in these animals.

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Various extraction procedures have been employed for the microbiological determination of vitamin B_<12> in the past. In view of the toxicity of sodium acetate to Ochromonas malhamensis, comparative investigations were made on the liberating procedures of vitamin B_<12> from natural materials using Escherichia coli, Lactobacillus leichmannii and Ochromonas malhamensis as test organisms. The results indicate that KCN method seems to be most suitable for the extraction of vitamin B_<12>, since the highest vitamin B_<12> values were obtained in the determination with any test organism used.

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Investigations were made on the causes of and countermeasures for high blank phenomenon in the microbiological determination of vittamin B_<12> with Lactobacillus leichmannii. One of the main causes of the phenomenon seems to be the change of requirement for vitamin B_<12> due to the mutation of the test organism during the stock culture, since a considerable destruction of the vitamin was recognized in the liver extract added to the stock culture medium during storage. The addition of a large amount of vitamin B_<12> to inoculum medium also resulted in high blank due to carrying over of the vitamin. It is desirable to use liver extract as fresh as possible for the stock culture medium and to add less than 0.5 mμg/cc of the vitamin to the inoculum medium.

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A test for collecting bacterial cell fraction of high vitamin B_<12> content was conducted using Sharples super centrifuge in the night soil treatment plant at Kotoura in Okayama Prefecture. The yield of bacterial cell fraction after 60 minutes operation averaged 480 g (15% moisture), corresponding to 0.1% on a dry basis toward the amount of the supernatant treated. Vitamin B_<12> content in 28 kinds of the samples determined with E. coli and Ochromonas malhamensis averaged 24.5 and 16.5μg/g, respectively. That is to say, about 70% of vitamin B_<12> activity obtained might be due to that active for animals. At any rate, further investigation is desirable on the collection methods.

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It was reported by Higashi that hypervitaminosis A tended to be overestimated since oxidized or polymerized products of vitamin A might be able to cause hypervitaminosis. This paper presents the investigation on hypervitaminosis A in rats using synthetic crystalline vitamin A acetate. The result indicated that the hypervitaminosis was recognized in an administration of 10,000 I.U. of the vitamin per day, and a severe disorder in 30,000 I.U.. The effect of the presence of vitamin E on the hypervitaminosis was not recognized.

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Investigations on the relationship between the amount of fat in diet and hypervitaminosis A in rats showed that more severe disorder was recognized in low fat diet than in high fat diet. It seems that the degree of the hypervitaminosis not only depends upon the amount of vitamin A absorbed, but also to the additive effect of nutritional deficiency such as that of essential fatty acids.

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The effect of 1-amino-2-ribitylamino-4,5-dimethylbenzene (diamine derivative) on the riboflavin formation by various intestinal bacteria was observed for the purpose of studying the mechanism of riboflavin biosynthesis. (1) Diamine derivative was effective in the formation of riboflavn. The highest formation was found when, glycine and urea were added to diamine derivative. (2) The riboflavin formation by diamine derivative was successful when washed cells of Escherichia coli communior were used, but not when the homogenate and cell free extract of the bacteria were used. (3) The ratio of FMN and free riboflavin to total riboflavin was generallly high in the addition of diamine derivative.

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The following results were obtained by observations on the content of riboflavin and other fluorescent substances of feces in men loaded with riboflavin. (1) When an excessive amount of riboflavin was loaded to men once a day for 5 days, the riboflavin content of feces increased remarkably on the 1st day, but it underwent no further changes. (2) A violet fluorescent substance having Rf 0.41 appeared always in human feces. It is likely that this violet fluorescent substance is an intermediate substance of riboflavin formation.

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The following results were found in shaking culture of Eremothecium ashbyii for 6 days. (1) The riboflavin content of the mycelium increased to a maximum on the 4th day, and it decreased later. (2) A blueish green fluorescent substance having Rf 0.17 was observed in both broth and mycelium. With the growth of the fungus, its fluorescence became stronger.

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The total content of riboflavin and the ratio of its 3 types were investigated on several kinds of shell-fish. The investigation was made with the whole body, internal organs, shell adductor and other muscles. The riboflavin content was determined by the lumiflavin fluorescence method and 3 types (FAD, FMN and free riboflavin) rotio by paper chromatography. (1) The riboflavin content of internal organs was higher than that in shell adductor and muscles. The vitamin content of the internal organs was higher in shell-fishes with a single shell, such as ear-shell, top-shell and river-snail, than in those with double shells. (2) Among the 3 types of riboflavin, FAD occupied the most part and FMN and free form followed in order of amount. In the shell adductor no free riboflavin was seen at all. But in the internal organs of ear-shell, top-shell and river-snail free riboflavin was the highest, followed by FMN and FAD.

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Present experiment was conducted to verify the interrelation between the thiaminase activities of human feces and the presence of "thiaminase bacteria". Frequency of feces showing positive thiaminase activity increased significantly when the activity test was performed in the presence of pyridine. Eight strains of "thiaminase bacteria" isolated from 330 samples of feces belonged to Bacillus thiaminolyticus Matsukawa et Misawa, one to Bacillus aneurinolyticus Kimura et Aoyama and 5 to Clostridium thiaminolyticum Kimura et Liao. The author could not always isolate thiaminase bacteria from feces showing thiaminase activity, nevertheless the frequency of isolation from feces having thiaminase activity was significantly higher than that of isolation from feces having no activity.

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From the feces of 341 human beings, the authors suceeded in isolating one strain of Bacillus aneurinolyticus and 30 strains of Bacillus thiaminolyticus. Studies of the morphological and biological properties of these bacteria led us to the conclusion that the result was almost the same as heretofore. The thiamine level of blood in 9 cases was estimated.

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Ten strains of Clostridium thiaminolyticum have been isolated from the feces of 125 human beings. Their morphological and biological properties, as well as the decomposition of carbohydrates were studied, comparing with those of Clostridium thiaminolyticum Kimura et Liao.

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As previously reported, pyrimidine derivatives of the toxopyrimidine group and substances of the carbonyl reagent group, with a totally different structure, induce the so-called running fit in the mouse. Studies were therefore conducted on the interrelationship between the two groups. Acute toxicity tests in the mouse showed that a combination of the two, in doses which when administered alone will not induce the running fit, results in an intensified seizure. The results suggest that there is a common mechanism of action for inducing the runnig fit.

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The action of 2-methyl-4-amino-5-hydroxymethylpyrimidine on the brain enzyme system, that is, glutamic decarboxylasse, γ-aminobutyric-α-ketoglutaric transaminase and the coenzyme supplying system, pyridoxal-kinase, was studied in vitro. Brain glutamic decarboxylase is not inhibited by 2-methyl-4-amino-5-hydroxymethylpyrimidine in its original form but is inhibited by the phosphorylated form. The γ-aminobutyric-α-ketoglutaric transaminase, however, is not affected by either form. It was also found that 2-methyl-4-amino-5-hydroxymethylpyrimidine inhibits the phosphorylation of pyridoxal.

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The action of an agent belonging to the atoxopyrimidine group, 2-methyl-4-amino-5-amino-methylpyrimidine, on the enzyme system of the brain related to the occurrence of the running fit, (glutamic decarboxylase, γ-aminobutyric-α-ketoglutaric transaminase and pyridoxal-kinase) was studied. It was found that all of these enzymes are inhibited and the inhibition of the former two is restored by pyridoxal-phosphate. Inhibition of the former two is intensified by preincubation but in this case, recovery was observed only with γ-aminobutyric-α-ketoglutaric transaminase.

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In an attempt to clarify the running fit from the standpoint of brain metabolism, the enzyme system involved in the metabolic pathway from glutamic acid to γ-aminobutyric acid and then to succinic semialdehyde in the brain was studied. For inducing the running fit, 2-methyl-4-amino-5-hydroxymethylpyrimidine and thiosemicarbazide were used and pyridoxine and 2-methyl-4-amino-5-aminomethylpyrimidine were used for the suppression. It was found that at the time of the running fit, glutamic-decarboxylase was inhibited whereas when the seizure was suppressed, there is no inhibition. There was no inhibition of the γ-aminobutyric-α-ketoglutaric-transaminase. The findings suggest that γ-aminobutyric acid is decreased during the running fit.

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It has been shown that isonicotinic hydrazide (INAH) induces the so-called running fit and this seizure is not suppressed by vitamin B_6. On the contrary, the administration of vitamin B_6 shortens the time interval of onset of the seizure. The action of INAH on the enzyme system involved in the running fit was studied. Results clarifying the effect of INAH on the brain enzyme system and the intensification of the seizure by vitamin B_6 were obtained.

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Brain γ-aminobutyric acid was examined during the running fit. It is reduced during the 2-methyl-4-amino-5-hydroxymethylpyrimidine induced seizure and this reduction is already apparent prior to the onset of the seizure so is not a result of the running fit. No reduction is found when the seizure is suppressed by the concomitant administration of pyridoxine or 2-methyl-4-amino-5-aminomethylpyrimidine. The γ-aminobutyric acid is also markedly reduced in the isonicotinic acid hydrazide induced running fit. When pyridoxine is administered at the same time, the seizure occurs earlier and the reduction in γ-aminobutyric acid also becomes apparent sooner. It is suggested that the cause of the running fit and the reduction in γ-aminobutyric acid are closely related.

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Experiments were conducted on the suppression of the running fit by γ-aminobutyric acid and β-hydroxy-γ-aminobutyric acid. The seizure was not prevented by γ-aminobutyric acid. It is believed that the γ-aminobutyric acid which is administered does not reach the brain effectively. The seizure was regularly inhibited by β-hydroxy-γ-aminobutyric acid. It is suggested that γ-aminobutyric acid is oxidized to β-hydroxy-γ-aminobutyric acid in the brain.

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