The cell renewal rate of small intestinal mucosa was examined, based on the P³² incorporation into DNA, in the groups of acute and chronic riboflavin deficiency, acute anemia by venesection and chronic iron deficiency in rats and the following findings were obtained.
1. In normal animals P³² incorporation into DNA in the epitheliar cells of the small intestinal mucosa arrived the maximal level about 48 hours after the injection, suggesting the complete replacement of the epitheliar cells of the mucosa in about 48 hours.
2. In both acute and chronic riboflavin deficiencies, the renewal rate of the epitheliar cells of the small intestinal mucosa did not decrease. In chronic riboflavin deficiency it was rather significantly increased.
3. The cell renewal rate was increased in acute anemia by venesection, whereas it was remarkably decreased in chronic iron deficiency.

View full abstract

1. Isolation and confirmation of 2′-deoxyinosine and related compounds which appeared in the reaction mixture were made by two-dimensional ascending paper chromatography, and each spot was applied to bioautography, autoradiography and chromatoscanning. The recoveries of the standard compounds previously added to the reaction mixture were satisfactory.
2. Formation of 2′-deoxyinosine-8-C¹⁴ from inosine-8-C¹⁴ In the presence of vitamin B₁₂ was tested by two-dimensional paper chromatography, and paper chromatoscanning, and the labeled deoxyinosine was detected in the amount of 8% of the added C¹⁴.
3. Using the similar method, the incorporation of the labeled carbon of hypoxanthine-8-C¹⁴ or D-ribose-1-C¹⁴ in deoxyinosine was studied. In the presence of hypoxanthine-8-C¹⁴, D-ribose and CN-B₁₂, the labeled carbon was found in the deoxyinosine fraction by paper chromatoscanning in the amount of 2.7% of the added C¹⁴, but in the presence of hypoxanthine, D-ribose-1-C¹⁴ and CN-B₁₂, the incorporation of the labeled carbon into deoxyinosine was scarcely detected.

View full abstract

With the use of a highly sensitive gas chromatograph equipped with hydrogen ionization detector, separation and determination of α-, γ- and δ-tocopherols were carried out. Following acetylation of the analogues, they were injected into the gas chromatograph in amounts of 5μl of 1-3% acetone solution after addition of squalene as the internal standard. Peak area was determined with a half value width methed. A rectilinear relationship between the peak area and weight was obtained in α-, γ- and δ-compounds within the range of 20-100μg. Recovery was 96.0-103.0%. Separation and determination of tocopherol analogues contained in the natural concentrate were easily carried out.
Although colorimetry according to the Nationnl Formulary gave similar values of total vitamin, higher values of α-tocopherol were always obtained with colorimetry than those determined by gas chromatography, probably indicating a defect in the determination of non-α-compound in colorimetry.

View full abstract

Various antioxidants were developed on silica gel G and polyamide thin layer plates using chloroform, methanol, and benzene as the developing solvents. Although silica gel G generally gave a better separation, a polyamide plate developed with methanol was more satisfactory for separating isoamylgallate, propylgallate and nordihydroguaiaretic acid.
In order to extract these antioxidants from vitamin A oil and vegetable oil, the compounds were extracted with an equal volume of methanol, and directly developed on a silica gel G or polyamide plate with petroleum ether, benzene, chloroform, and methanol respectively. Separation and detection of as little as 0.1μg of butylhydroxyanisol and dibutylhydroxytoluene and 0.5μg of isoamylgallate and nordihydroguaiaretic acid thus became possible without significant inhibition of the contaminants.

View full abstract

When the benzene solution of vitamin K₁ was irradiated with ultraviolet light many colored and fluorescent degradation products were produced as detected in thin layer chromatography according to the following scheme.
The spot 2 substance is the compound produced by the change of the phytyl double bond but the latter bond is not required for the formation of the spot 3 and 4 substances. The spot 3 substance is identified as phthiocol and is one of the final degradation products.

View full abstract

When vitamin A acid reacted with SbCl₃ in chloroform, a purple red color having the maximum absorbance at 573mμ developed. The maximum gradually moved to 470mμ. Upon stopping the reaction by addition of water at the maximum color intensity at 573mμ, a yellow fluorescent substance (I) was mainly formed. Similar treatment at the maximum color intensity at 470mμ gave rise to a blue fluorescent substance (II).
It was revealed from the findings of thin layer chromatography, ultraviolet absorption spectrum, SbCl₃ reaction and the color of fluorescence that the substance I was changed to the substance II by the treatment with SbCl₃. Reaction of vitamin A acid with HCl or acid clay gave rise to substance I but not to substance II.

View full abstract

For proving the formation of protein alkylmercaptan mixed disulfide by the reaction of asymmetric disulfide-type thiamines with the SH-groups of proteins, investigation was made by the method of microassay using HgCl₂ and dizithon and the following results were obtained.
1. Free thiamine and bound benzyl mercaptan were produced by the incubation of thiamine benzyl disulfide with heat-denatured egg albumin at neutral pH regions but it was not the case with albumin having no reactive SH-groups.
2. Similar complexes were formed by the reaction with urea-denatured ovalbumin, bovine serum albumin or β-lactoglobulin. The ratio of the bound mercaptan to free thiamine varied somewhat with the ratio of the reactants (disulfide/protein) and the kind of proteins. It was similar to the ratio of bound thiamine to free thiamine in the reaction of symmetric disulfide-type thiamine, Formation of bound thiamine was not observed in all the proteins.
3. From these results it was proved that asymmetric disulfide-type thiamine reacted with protein-SH mainly according to the Equation a. The difference of the reaction mechanism between asymmetric and symmetric disulfide-type thiamines were considered from the view point of thiol-disulfide exchange reaction.

View full abstract

Microassay procedure of protein-SH groups and low-molecular SH-groups using thiamine disulfide derivatives was investigated and following results were obtained.
1. By estimating by thiochrome method the thiamine formed in the reaction of 0.1-0.2μmole of thiol derivatives with 5μmoles of thiamine disulfide in the presence of 10-100μmoles of EDTA (5-10ml in final volume) at pH 7-8 for 5-30 minutes at 30°, SH-groups can be safely determined. It is also applicable even at pH 6 to 9.
2. The sensitivity of this method is markedly high and microassay of as little as 0.002μmole of thiol derivatives is possible. Amino acids other than cysteine neither react with thiamine disulfide under the assay condition nor inhibit the determination of cysteine. The values of cysteine, glutathione β-mercaptoethylamine, thioglycollic acid and 2, 3-dimercapto-1-propanol agreed well with those determined by Boyer's PCMB method.
3. When this method was applied to several protein-SH groups, most of them failed to be determined in the native form, but the values increased after denatura tion with urea, sodium dodecylsulfate, guanidine, heat or alkali, finally reaching the level almost in agreement with the values of PCMB method.
4. The utilization of symmetric and asymmetric thiamine disulfide derivatives other than thiamine disulfie was also investigated and O-benzoyl, O-propionyl, O-butyzyl, thiamine disulfides, as well as thiamine benzyl and thiamine propyl disulfides were found to be applicable to this method. The asymmetric thiamine disulfide derivatives were found to have remarkably high reactivity with native serum albumin-SH groups.
5. Based on these results, the characteristic of this method were discussed and the standard assay procedure was described.

View full abstract

1. When 10μmoles of thiamine disulfide (TDS) or O-benzoylthiamine disulfide was added to 0.2μmoles of SH-blocked urea-denatured bovine serum albumin and the mixture was incubated at pH 9.0 and 37°, the formation of protein-thiamine mixed disulfide, at most 4.5 moles per mole protein, was observed.
2. This reaction was almost completely inhibited by SH-reagents, such as p-chloromercuribenzoate (PCMB) and N-ethylmaleimide, about 1/10 mole of TDS, whereas it was markedly accelerated by the addition of thiamine, corresponding to 1/50 to 1/10 mole of TDS.
3. In the reaction with iodine-oxidized egg albumin, the formation of similar compound and its inhibition by PCMB were observed.
4. From these results, it was assumed that protein-thiamine mixed disulfides were formed from protein SS-groups and symmetric disulfide-type thiamines by the similar disulfide-disulfide exchange reaction of low molecules catalyzed by a trace amount of thiols.
5. Asymmetric disulfide-type thiamines such as thiamine benzyl disulfide and thiamine propyl disulfide were found to produce both protein-bound thiamine and protein-bound alkylmercaptan under the same conditions.
6. Considering the results of this paper and those of the previous findings, it was concluded that there are three types of exchange reaction between thiamine derivatives and proteins.

View full abstract

A simple assay method of methylmethionine sulfonium derivative was established utilizing the precipitation reaction with phosphotungstic acid and the coloration with potassium 1, 2-naphthocluinone-4-sulfonate. This method could satisfactorily be applied for the determination in rather complicated, mixed preparations.

View full abstract

Hexa-, tetra- and mono- fatty acid esters of myoinsitol were synthesized by various methods, e.g., by heating myoinositol or its 1, 2-isopropiridene derivatives with acid anhydrides of various fatty acids, or by ester exchanging reaction with methyl esters of fatty acids under coexistence of sodium methoxide as catalyst.
Of the fatty acid esters of myolnosltol prepared, hexaacetate, hexapropionate and hexabutyrate showed similar growth promoting effect as free myoinositol upon young rats, whereas hexavalerate, tetrabutyrate, monooleate and monolinoleate showed no influence, and some of them showed rather inhibitory effect. These three esters that possess growth promoting effect in rats showed beneficial effect for the prevention of fatty liver in the Handler's experimental systems. The lipotropic action of myoinositol hexanicotinate was quite obscure.
Some probabilities for the application of these fatty acid esters of myoinositol were briefly discussed in connection with their physicochemical properties.

View full abstract