The activities of thiamine disulfide (TDS) and its derivatives on the growth of Lactobacillus fermenti 36 and of Lactobacillus viridescens S38A were was investigated under various conditions. The activities of TDS and its cetylsulfate (TDS-CS) to both the organisms in the medium without cysteine or cystine were lower than that of thiamine hydrochloride. Under the same conditions, O-propionylthiamine disulfide, O-acetyl-, O-furoyl- and O-benzoylthiamine disulfides showed only a slight activity. However, the activities of TDS and TDS-CS became higher than that of thiamine when the medium contained cysteine or cystine, especially the derivatives were autoclaved with the medium. The other derivatives showed 10-80 per cent activity of thiamine under the same condition.

View full abstract

General pharmacological properties and toxicity of O-benzoylthiamine disulfide (BTDS) were studied and the following findings were obtained.
1. General Pharmacological Action
In the respiratory and circuratory systems BTDS behaved qualitatively similarly to thiamine hydrochloride, but quantitatively the former showed a little stronger activity. A dose of 5mg/kg BTDS resulted in the gradual fall of the blood pressure of the rabbit, somewhat more marked than 5mg/kg thiamine hydrochloride. However, no effect was detected on the respiration of the animal. Upon the isolated toad heart, 10^-6 M BTDS showed a slight inhibitory action and at concentrations higher than 10^-4 M the derivative inhibited more markedly the amplitude of the hart beat. In the case of thiamine hydrochloride, similar effect was seen at 10^-5 M, and it was more marked at higher concentrations.
Upon smooth muscle organs, the mode of action of BTDS was quantitatively the same as thiamine hydrochloride. BTDS showed scarcely any vasoconstrictive effect on the rabbit auricular vessels similarily to thiamine hydrochloride. On the motility of the isolated rabbit intestine BTDS revealed inhibitory action, lowering the tonus at concentrations higher than 10^-4 M, whereas thiamine hydrochloride did not show any effect at the concentration of 10^-3 M. In the rabbit uterus in situ, normal movement was observed without any effect of 5mg/kg BTDS or thiamine hydrochloride given intravenously.
On the contrary, Tsunoo (3) had reported that the rabbit respiration was somewhat accerelated, the blood pressure rose a little, auricular vessel was dilated and the tonus of the isolated rabbit uterus was increased following an intravenous administration of BTDS. The contradiction between the findings of Tsunoo and of the author is possibly due to the difference in the dose of BTDS. Both have the same view that BTDS has a little stronger pharmacological action than thiamine hydrochloride.
2. Toxicity
The LD₅₀ values of BTDS for mice were found to be definitely high in intravenous, intraperitoneal and oral administrations in comparison with thiamine hydrochloride.
Following the oral administration of a large dose of BTDS to rats for 6 months, a significant growth promotion was observed without marked changes in blood cell counts, hemoglobin contents and leucocyte distributions, and neither change nor damage was observed pathologically.

View full abstract

The action of the thiaminases produced by Bacillus thiaminolyticus Matsukawa et Misawa and Bacillus aneurinolyticus Kimura et Aoyama on various thiamine disulfide derivatives were investigated, and thiamine disulfide cetylsulfate, O-benzoylthiamine disulfide and O-acetylthiamine disulfide were found to be definitely resistant to the enzymes.

View full abstract

1. At high concentrations, pyridoxal phosphate inhibited the blood coagulation both in vivo and in vitro. This inhibitory action is not physiological.
2. At very low concentrations, pyridoxal phosphate activated the fibrinolytic activity both in vitro and in vivo.
3. It was demonstrated from the absorption spectra that ε-aminocaproic acid combined with pyridoxal phosphate in vitro.

View full abstract

1. Activity tests of various dispersoids on the intestinal movement, using isolated segments of the small intestine of the rat under well-controlled experimental conditions, demonstrated that free vitamin A alcohol showed excellent activity on the intestinal movement, but the acetate was always inactive. In the case of vitamin A palmitate, the diminished excursion was observed at the initial stage, while the excursion recovered to normal after a few minutes. It seems to be possible that the results are due to the presence of vitamin A palmitate-esterase.
2. Vitamin A aldehyde induced a little larger excursion, whereas vitamin A acid was completely inhibitive. Vitamin A alcohol-vitamin A acid-ester showed a diminished excursion at the initial stage, but after a few minutes increased rate of the intestinal movement was seen.
3. Natural vitamin A alcohol concentrate always indicated excellent action on the rat intestinal movement.
4. No activity was observed with β-carotene, whereas vitamin A alcohol derived from β-carotene by incubation with fresh rat intestinal tissue for 90 minutes always indicated active results.
5. These findings seem to suggest that free vitamin A alcohol may be linked to the intermediate source of hydrogen transfer for the muscular work in the intestinal tissue.

View full abstract

1. After subcutaneous injections, the recoveries of thiamine were generally greater than that after oral administrations.
2. The duration of the vitamin excretion was generally longer after subcutaneous injection than after oral administration.
3. Of the thiamine derivatives, thioctic acid-thiamine disulfide administration resulted in less elimination as thiamine in the urine. Benzoylthiamine disulfide continued longer in urinary excretion.

View full abstract

1. Paper chromatographic separation (ascending method) of the mixture of L-ascorbic and D-araboascorbic acids was carried out by using the papers impregnated with 3% metaphosphoric acid and water-saturated methyl ethyl ketone as a solvent system. 6 to 40 μg per spot of each isomer could be clearly distinguished by spraying 0.013% indophenol solution, the difference in R_F values for both acids being close to 0.1. Developing temperature of 20 to 25° and developing time of 5 to 5.5 hours were recommended for the separation.
2. Effects of moisture and partition liquids in the stationary phase were also studied. By increasing the concentration of glycerol in 3% metaphosphoric acid, the locations of both the isomers were varied without change of separating ratio. By using this procedure, the stability of the R_F values for the acids was increased. This technique may be effective for separating the substances from natural products which contain some impurities having the same R_F values as these acids.
3. Influences of some carbohydrates and their derivatives on the separation of the isomers were studied at various temperatures. L-Ascorbic and D-araboascorbic acids were shown to be completely separated from carbohydrates even if the isomers existed in the dehydro-forms.
4. After separation, the isomers were estimated by 2, 4-dinitrophenyl hydrazine method. Experimental results were treated by analysis of linear regression and 95% fiducial limits of regression lines were determined. 95% fiducial limits of regression lines were ±3 to 4μg for both compounds at a range of 10 to 50μg and ±2 to 3μg for 4 to 10μg.

View full abstract

1. Heterogeneity of transaminase activities was proved by electrophoretical separation of the homogenates of the heart, kidney, liver, skeletal muscle, brain, pancreas and intestine of the rat. Four peaks of transaminase activities were found in the liver, three peaks in the heart, kidney, skeletal muscle, brain, pancreas and intestine. Each tissue exhibited a similar distribution of trasaminase activities.
2. The activity peak electrophoretically separated had the same mobility as the peak in the liver having the greatest transaminase activity. The acetone-dried powder extracts showed higher transaminase (GOT₁, GOT₂ and GPT₂) activities than homogenate extracts.
3. The enzymes isolated from rat tissue-acetone powders were shown to differ in sensitivity to inhibition by heating, trypsin, lipase and isonicotinic acid hydrazide.

View full abstract

A purification method of kynurenine transaminase is described. The steps consist of pH 5 treatment, ammonium sulfate fractionation, heating at 60° for 5 minutes, and hydroxyl apatite column chromatography. About 200-fold purification has been achieved starting from the supernatant of rat kidney homogenate.
α-Ketoglutarate and pyridoxal phosphate have been found to afford considerable protection of kynurenine transaminase against thermal denaturation.
Spectrophotometric measurements on the purified enzyme revealed the absorption bands at 280 and 410 mμ. This enzyme has an optimum at approximately pH 6.8.
The effect of adding various reagents on the activity of the purified enzyme has been determined.

View full abstract

For the purpose of investigating the non-cyano form of vitamin B₁₂ occurring in nature, studies were conducted on the effect of various conditions of extraction and sterilization on the microbiological determinations of the vitamin using a bacterial preparation commercially available for supplementing the vitamin to the animal feed and the following results were obtained.
1. Differing from the active sludge and the methane fermentation bacterial fraction, a microbiologically active extract, equal in activity to the extract with hot aqueous cyanide, was obtained by simple water extraction at room temperature.
2. The non-cyano B₁₂ obtained by water extraction at room temperature showed the activity equal to cyanocobalamin with the Lactobacillus leichmannii method described in U.S.P. XV, but an extremely low activity with the Ochromonas malhamensis method using the cyanide-free Ford medium.
3. Scarcely any decrease in activity was found with the Ochromonas method, when stabilizing agents such as ascorbic acid, thioglycolic acid or KCN is added or when the germfree extract obtained by filtration without heat sterilization was added aseptically. It suggests that the non-cyano B₁₂ Per se in the sample is not poorly active to Ochromonas, but it is degraded in the course of heat sterilization with the Ford medium.

View full abstract

Investigations were carried out in an attempt to establish an accurate method for separation and determination of non-cyano forms of B₁₂. A bacterial preparation was extracted with water and the extract was subjected to paper electrophoresis using a CN-free acidic electrolyte solution for a relatively short time (2 hours). Thus the separation without decomposition of the vitamin was successful. Further, microbiological determination of the sections which had been cut out of the filter paper and extracted with cyanide-containing water to convert all the non-cyano forms to cyano forms, were carried out using Ochromonas as the test organism. Thus the fractional determinations of various B₁₂ factors active to Ochromonas, having various groups coordinated to the cobalt atom, were achieved. Using this method the bacterial preparation used was found to contain three forms of B₁₂, of which about 50% was CN form, about 30% OH form and the rest a protein-bound form.

View full abstract

1. Stoichiometric interconversion of glutamate and aspartate by the coupling reactions between GOT_M and TCA cycle takes place in the isolated mitochondrial system.
2. Significance of the GOT_M cycle for amino acid metabolism may concern with nonessential amino acid catabolism and anabolism and with the effective pathway, through which the amino group of glutamate turns into urea without liberation of ammonia.
3. Elementary reactions of GOT_M strongly occur in mitochondria, but the physiological significance is not known.

View full abstract