1. Thiamine propyl disulfide (TPD) is rapidly reduced in blood even at low temperature while it is relatively stable in a metaphosphoric acid-treated solution of blood.
2. The content of TPD in the mesenteric venous blood was maximum 1 minute after infusion into a ligated intestine, showing 25% absorption by a method of TPD determination. The value decreased with time.
3. The existance of TPD in the mesenteric venous blood was demonstrated bioautographically using a thiamine-less mutant (70-23) of E. coli.

View full abstract

1. In the present work, several analogues of 5, 6-dimethyl benzimidazolyl cobamide coenzyme (DBCC), containing hydroxyalkyl group linked to the cobalt atom, were synthesized as the possible intermediates in the diol-dehydrase system on the assumption of active participation of the cobalt atom, and their properties, above all, behaviors in photolysis and in the enzymatic system were examined.
2. Co-β, γ-dihydroxypropyl-, Co-β-hydroxypropyl-, and Co-hydroxyethylcobalamins were prepared by reacting glycerol α-monochlorohydrin, propylene bromohydrin and ethylene chlorohydrin with vitamin B_12s. Co-β, γ-dihydroxypropylcobinamde was also synthesized by the reaction of glycerol α-monochlorohydrin with a reduced product of monocyanomonoaquocobinamide with NaBH₄.
3. Absorption spectra of the three hydroxyalkylcobalamins were similar to those of DBCC or Co-alkylcobalamins, and the spectrum of the hydroxyalkylcobinamide was analogous to those of cobinamide coenzyme or Co-alkylcobinamide. These four compounds, alike cobamide- and cobinamde coenzymes, were very unstable in light and easily decomposed to hydroxo (or aquo) forms. When they were allowed to stand in the dark in 0.017 M KCN solution, they were converted only partly to dicyano forms like the alkyl derivatives. Paper chromatographic behavior showed their higher hydrophilicity than alkyl derivatives as expected.
4. Hydroxyalkylcobalamin, for example Co-β, γ-dihydroxypropyl cobalamin, showed an almost equal activity on vitamin B₁₂-requring E. coli. However, this compound did not exhibit the coenzyme activity, and, moreover, exerted an inhibitory effect, like the alkyl derivatives, in the diol-dehydrase system. From the result it would be concluded that the hydroxyalkyl derivatives of DBCC so far tested are not the active intermediate of the diol-deoxyaldehyde conversion system.
5. Co-β, γ-dihydroxypropylcobalamin was irradiated (a) under bubbling of air, (b) in the presence of dissolved oxygen, (c) in a nitrogen atmosphere, (d) in the presence of NaBH₄. The photolysis products formed from the dihydroxypropyl group were as follows: (a) glyceric acid. (b) glyceraldehyde and glycerol, (c) glycerol and a small amount of an unidentified substance and, (d) glycerol. The decomposition routes of this compound are considered to be analogous to those of DBCC. The formation of glycerol is not consistent with the formation of 8, 5′-cycloadenosine from DBCC. It is similar to the formation of methanol in the photochemical decomposition of Co-methylcobalamin reported by Weissbach et al. The reaction mechanism may be interpreted on the formation of vitamin B_12r and dihydroxypropyl free radical by the homolytic fission of cobalt-carbon bond.

View full abstract

1. Reaction of α-lipoamide with potassium cyanide in dilute ethanol was investigated. It was thereby found that α-lipoamide was converted to 5-(2-thiacyclobutyl) valeramide almost quantitatively with loss of sulfur.
2. 5-(2-thiacyclobutyl) valeramide has many advantageous points in its gaschromato-graphic behavior, such as sensitivity, reproducibility and retention time, as compared with those of α-lipoamide.
The conversion of α-lipoamde to 5-(2-thiacyclobutyl) valeramide, therefore, will be a very useful means for the determination of α-lipoamide.

View full abstract

1. Heterogeneity of xylulose reductase activities was proved by electrophoresis on cellulose acetate of rat and human liver or kidney homogenates. Two separable forms of xylulose reductase activities were found in these tissues.
2. One is associated with the mitochondria and the other is mainly in the supernatant fraction of the cells.
3. An aqueous extracts of acetone powder of rat and human liver showed also two xylulose reductase activities.
4. The enzyme of one fraction was easily inhibited by trypsin.

View full abstract

As the fundamental studies on the quantitative determination of pyridoxal phosphate, its separation from other B₆ analogues was investigated by using thin layer chromatography and cellulose ion exchanger column chromatography and the following findings were obtained.
1. The fluorescence and the Gibbs coloration of pyridoxal phosphate, pyridoxine, pyridoxal, pyridoxamine, pyridoxamine phosphate and pyridoxine phosphate on the thin layer were individually different according to the kinds of thin layer adsorbents.
2. The limit concentration for detection of pyridoxal phosphate and other B₆ analogues on the thin layer was about 10^-4M. In the case of real development, the limit concentration of pyridoxal phosphate may be greater than 10^-3M.
3. Dioxane-water (7:3) was found to be the suitable solvent for separating pyridoxal phosphate from other B₆ analogues on the cellulose thin layer. But if the reproducibility of R_f value is exactly requested, the rigid control of the experimental condition is needed.
4. The recovery of the pyridoxal phosphate spotted on cellulose thin layer was 96.9%. Thin lager chromatography is thought to be the method to be tried once for separation of pyridoxal phosphate from other B₆ analogues. But when its concentration is less than 10^-4M, concentrating operation of the sample seems to be necessary.
5. DEAF-, TEAS-, SM- and SE-cellulose ion exchanger were preliminaly investigated for the separation of pyridoxal phosphate from other B₆ analogues. It was found that 0.01 N HCI was suitable for separation in the case of DEAE cellulose column, 0.01M acetate buffer (pH 4.4) in the case of TEAE-cellulose column, 0.01N acetic acid and 0.01M acetate buffer (pH 4.7) in the case of SM-cellulose column and 0.001M acetate buffer (pH 4.7) in the case of SE-cellulose column.
6. The recovery of pyridoxal phosphate was 90 to 98% in the case of TEAE-cellulose column and 97 to 99% in the case of DEAF-cellulose column. Subsequently, ion exchanger column chromatography is considered to be able to separate pyridoxal phosphate from other B₆ analogues.

View full abstract

1. The treatment of diabetic neuropathy with daily oral dose of 10mg of CoQ₇ for 3 to 13 weeks was made and obtained considerable relief from neurological subjective symptoms such as pain and paresthesia of extremities, and calftenderness as well as improvement in the abnormal threshold of vibratory perception on fingers and toes.
2. Impaired metabolism of α-keto acids observed in diabetics, as measured by the increase of urinary pyruvate and α-ketoglutarate after intravenous fructose loading, was improved considerably after oral administration of CoQ₇.

View full abstract

Various O, S-disubstituted cholic acid esters of thiol type thiamine (I-V) were synthesized. These compounds were negative for thiochrome reaction but readily regenerate thiamine by treatment with alkali. Biological tests for these compounds are now being undertaken.

View full abstract

1. On non-anesthetized dogs provided with the normal, decentralized and aganglionic Thiry-Vella jejunal loops the effect of thiamine tetrahydrofurfuryldisulfide (TTFD) upon the intestinal motility was studied.
2. On the normal and decentralized loops the intravenous administration of 1.5mg/kg of TTFD produced a slight rise of tone and a remarkable increase of the amplitude of rhythmic contractions for about 25 minutes.
3. In the animal anesthetized with 25mg/kg of pentobarbital sodium the drug also increased the motility of the normal and decentralized loops as remarkably as in the non-anesthetized animal.
4. The drug exerted no action upon the aganglionic loop.
5. A small amount (1ml) of 5mg/kg TTFD solution which had been carefully introduced into the intestinal lumen, stimulated the mucosa to elicit the mucosal intrinsic reflex the effect of which consisted of an excitation above and inhibition below the stimulated spot.
6. The probable site of action of the drug was discussed.

View full abstract

1. Pyridoxine, thiamine tetrahydroxyfurfuryl disulfide and tocopherol were found to reduce hypercholesterolemia dietarily induced in rats.
2. Safflower oil also suppressed in some degree hypercholesterolemia but its action was enhanced by the combined use of pyridoxine.
3. Although administration of safflower oil enhanced fat deposition in the liver, it was reduced in some degree by the combined use of pyridoxine.

View full abstract

The thiaminase activity of the culture supernatant of clostridia received from many institutes in Japan was examined. Some of the cultures labelled as Cl. aerofoetidum, an organism related closely to Cl. bifermentans, Cl. botulinum, Cl. chauvoei, Cl. saprogenes, Cl. sporogenes, Cl. tetanomorphum and unclassified organisms were found to produce thiaminase I.

View full abstract

The new oxidized produucts of thiol-type thiamines, 2-(2′-methyl-4′-aminopyrimidyl-5′)-methylformamino-5-hydroxy-Δ²-pentenyl-3-sulfonic acid (thiaminic acid) and its O-benzoyl ester, were obtained by the oxidation of thiamine disulfide or its O-benzoyl ester with hydrogen peroxide. Thiamine alkyl disulfide derivatives, S-acyltype thiamine derivatives and cyanothiamine are also oxidized to thiaminic acid derivatives in similar oxidation reactions. However, no formation was found from thiamine alkylsulfide. Therefore, the thiol-type thiamine derivatives which are converted to thiamine by the reaction with sulfhydryl compounds such as cysteine or protein-SH, produce thiaminic acid derivatives under such oxidation reactions. Thiamine cannot take the thiol form in an acid medium and no thiaminic acid can be formed. Furthermore, by similar oxidation of several aryl thiamine disulfide derivatives or by acylation of thiaminic acid, several thiaminic acid derivatives were obtained. Thiaminic acid is an interesting compound which can be considered to occur in the body as an intermediate of thiamine metabolism and is assumed to be an effective form of so-called “active” thiamine derivatives.

View full abstract

In order to study more in detail the formation of thiaminic acids by the oxidation of thiol type thiamine derivatives, the oxidation condition of O-benzoylthiamine disulfide (BIDS) was investigated and the following results were obtained. In the oxidation with hydrogen peroxide, nearly the same yield was obtained in lower fatty acids such as acetic, formic, propionic or butyric acid and in its solution in water or various organic solvents. Further optimal condition was that more than 5 moles of H₂O₂ per mole of BTDS was allowed to react at about 40° for 2 to 4 days, whereby the yield was about 50%, showing that at least one mole of O-benzoylthiaminic acid (BTnA) was produced from one mole of BTDS. Furthermore, it was observed that BTnA was also obtained by H₂O₂ oxidation in an aqueous solution (pH 4-9) or by oxidation of bromine, permanganate or bichromate though in a fairly reduced yield.

View full abstract

The physicochemical properties of thiaminic acid (TnA) and O-benzoylthiaminic acid (BTnA) were investigated and the following results were obtained.
1. Each monohydrate crystal is relatively stable in drying in vacuo but by refluxing with ethanol, it is converted into anhydride.
2. Ultraviolet absorption spectra are similar to those of thiamine disulfide and O-benzoylthiamine disulfide, respectively. The spectra vary according to pH, owing to the resonance in the pyrimidine ring. Each compound has isosbestic point at about 273mμ.
3. Each compound is hardly soluble in organic solvents. Thiaminic acid is readily soluble in water, while BTnA is a little soluble at pH above the isoelectric point. However, it is hardly soluble in lower pH levels.
4. Both of them are almost negative to the Dragendorff reagent and also negative for thiochrome reaction even after treatment with cysteine or thiosulfate. On paper chromatogram they can be detected by tert-butyl hypochlorite method as a violet spot. The Rf values of TnA and BTnA with n-butanol-acetic acid-water are 0.25 and 0.54, respectively. These values are a little higher than the corresponding compounds, thiamine (0.17) or O-benzoylthiamine (0.5).
5. By paper electrophoresis, thiamine and O-benzoylthiamine migrate toward the cathode at pH 1.0-8.0, as a cationic form, while TnA and BTnA migrate toward the cathode at pH 1, do not move at pH 3.0-5.0, and toward the anode at pH sending its amphoteric character.
6. From the pontentiometric titration curve, the apparent dissociation index was determined at 25±1°. pK₁ (-SO₃H) was 2.2 (TnA) and 2.3 (BTnA), and pK₂ (-NH₃⁺) was 6.2 (TnA) and 6.1 (BTnA). The isoelectric points are both at pH 4.2.
7. By HCl-hydrolysis of TnA, formic acid and 2-methyl-4-amino-5-aminomethyl-pyrimidine were formed, whereas in the case of BTnA, benzoic acid was additionally formed.

View full abstract

Thiaminic acid (TnA) and its O-benzoyl derivative (BTnA) had no thiamine activity on love-birds. Oral administration of large dose, daily 600μg thiamine-equivalents, with or without thiamine (3μg daily), resulted in no significant difference in body weight and prolongation of life between the control and the given groups. This finding suggests that the compounds have no antithiamine activity in love-birds when given orally.
The toxicity was found to be very low among the compounds related to thiamine. The LD₅₀ of BTnA for mice by intravenous injection (pH 7.0, aqueous solution) was 1.87g/kg and that of TnA (pH 7.0, aqueous solution) over 4g/kg. No toxic effect was observed after either oral or intraperitoneal administration.

View full abstract