1. Thiamine-protein complex and non-bound disulfide-type thiamine are formed by the reaction of thiamine or O-benzoyl thiamine with the iodine-oxidized egg albumin at pH 7.3 or 9.0, and 37°. The thiamine bound with protein could not be separated from the protein by zone electrophoresis, dialysis at pH 3.0, deproteinization with metaphosphoric acid. However, free thiamine was easily liberated from the bound form by reduction with sodium thiosulfate or cysteine. These findings suggest that the compound is a protein-thiamine mixed disulfide produced by the interchange reaction of the thiol form of thiamine with the artificially produced SS-group of the oxidized protein.
2. The amount of the bound form is by far greater at pH 9.0 than at pH 7.3, possibly due to the difference in the amount of thiol-type thiamine.
3. In the reaction at the same pH level O-benzoyl thiamine was more reactive with oxidized protein than thiamine.
4. The amount of bound thiamine produced in the reaction with the oxidized egg albumin rises with the amount of iodine used for oxidation of the protein, up to about 5 equivalents of iodine per mole of egg albumin. Beyond that value, the bound form did not increase, taking about the constant value within the range of 6 to 18 equivalents.

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1. The amounts of protein-thiamine mixed disulfide produced by the reaction of thiamine or O-benzoyl thiamine (OBT) with oxidized egg albumin at various pH levels were determined. The formation of the bound form of thiamine was detected at pH about 7.0, increasing suddenly at pH above 8.2, and reaching a nearly constant value at pH above 10.0. The pH-curve was in good agreement with the curve of pH and formation of the thiol form, showing the parallelism between the production of the bound form and the concentration of the thiol type thiamine.
2. OBT showed already the reactivity at pH around 6.0, and more amounts were bound with protein than thiamine at any pH levels.
3. The equilibrium concentrations of the thiol form of thiamine and of OBT were compared in weakly alkaline range by measuring the anodic wave height of polarography. The anodic wave height of OBT was found to be far higher than that of thiamine, showing that OBT was more liable to take the thiol form. These findings suggest that the difference in the reactivities of thiamine and OBT in weakly alkaline range is mainly due to the difference in the formation of the thiol form.

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1. Urinary excretion of thiamine in 24 hours after intraperitoneal injection of benzenesulfonylthiamine disulfide (BSTDS), equivalent to 5mg of thiamine-HCl, to rats was about 3 per cent (as thiamine or benzenesulfonyl-thiamine). It is markedly low as compared with thiamine disulfide or thiamine propyl disulfide, suggesting the conversion of BSTDS to thiamine anhydride (TA) in the body. BSTDS was degraded nonenzymatically by rat liver homogenate and it was converted to TA by the reaction with thioglycolate. Its low urinary excretion is possibly due to the conversion of BSTDS to thiamne anhydride. However, thiamine anhydride or its sulfoxide could not be demonstrated as the urinary metabolite.
2. The thiamine activity of BSTDS was studied in rats by determining the increase of the body weight. Daily administration of BSTDS equivalent to 100μg of thiamine showed a significant increase but its administration equivalent to 10μg of thiamine was inffective. Oral administration resulted in more rapid rise of body weight than after intraperitoneal injection.

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1. Thiamine-8-(methyl-6-acethyl-dihydrothioctate)-disulfide hydrochloride (TATD) showed almost the same activity as thiamine or thiamine disulfide on L. fermenti in the culture medium containing cysteine, but inhibited the growth of the organism at 10^-2×1/4M. In the medium without cysteine the activity of TATD was reduced to one-tenth that of thiamine, i.e., about the same activity of thiamine disulfide but at concentrations above 10^-3×1/4M TATD inhibited the growth of the bacterium.
2. TATD showed the same activity as thiamine or thiamine disulfide to Kl. apiculata. It showed no inhibitory effect at high concentrations.
3. The biological activity of TATD seems to be due to its reduction into thiamine, and the inhibitory effect to be due to the compound per se. Since this inhibition is partially recoveredby addition of thiamine, TATD seems to be competitive against thiamine.

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Egg albumin oxidized with iodine reacts with the thiol form of thiamine or O-benzoylthiamine (OBT), forming a protein-thiamine mixed disulfide. The amount of the disulfide formation was markedly decreased by heat or urea denaturation before oxidation. The reactivity was greatest, when the SH-groups of the native protein was oxidized directly. Denaturation after oxidation did not cause any significant change in reactivity. When the SH-groups of the native protein was oxidized after denaturation, the reactivity was lowered with the degree of denaturation. The finding seems to be due to the decreased formation of the intramolecular -S-S-linkages in the protein molecule which are involved in the reaction with the thiol forms of thiamine derivatives owing to the denaturation prior to oxidation.

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Native or urea-denatured egg albumin was allowed to react with C₃H₇SSC₃H₇ (I) or C₃H₇SSOC₃H₇ (II) under a certain condition. The protein was precipitated by ethanol treatment and the unreacted I or II was completely removed by extracting the precipitate several times with hot ethanol. The protein fraction was reduced by cysteine, producing the free propylmercaptan. It is assumed to have been produced from the compound bound with the protein similarly to S-propylmercapto-cysteine. As the microdetermination of propylmercaptan was possible by utilizing dithizon method, the amount of the bound propylmercaptan under various contions was investigated and the following results were obtained.
1. The formation of the bound form of I was markedly increased by protein denaturation, producing also the free propylmercaptans. The formation of the bound form of II was somewhat increased after denaturation of the protein, but was observed significantly with the native protein. In this case the formation of free propylmercaptan was scarcely observed.
2. The reaction of I with the protein was markedly dependent on pH levels, rising definitely at pH levels above 7.0. On the other hand, the reaction of II was little affected by pH levels, showing almost a constant activity at pH range above 6.0.
3. The reactivity of I or II was increased with the rise of temperature and time.
4. When the SH-groups of the protein were oxidized with iodine or blocked by p-chloromercuribenzoate, the reactivity was remarkably reduced.
These findings suggest that the compound I or II, though the reaction mechanisms are different from each other, reacts with the SH-groups of the protein, forming a protein-bound compound like protein-SS-C₃H₇.

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It has been confirmed that the USP XVI method is the most desirable one for the determination of all-trans vitamin A and of a mixture of all-trans vitamin A and neovitamin A.

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The addition of 10^-2 to 10^-3M L-ascorbic acid to the reaction medium was found to greatly promote the white mustard myrosinase activity under the condition (pH 6.4), producing neither white turbidity nor hydrogen sulfide. The amount of the white turbidity and hydrogen sulfide produced on addition of ascorbic acid to the reaction mixture of myrosinase and sinigrin increased with the decrease of the pH value of the medium. Any of those materials was produced in a larger amount in the case of white mustard enzyme than in the case of mold enzyme. The material producing white turbidity was found to be composed mainly of protein. It may be concluded that ascobic acid is a specific activator of myrosinase.

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1. Urinary excretion of S³⁵ from the propylmercapto group after subcutaneous, intravenous or oral administration of TPD-S³⁵(outer) was studied and S³⁵ was shown to be excreted in the urine in large quantities (about 70% on the first day).
2. The majority of S³⁵ was excreted in the urine in the form of sulfate after administration of TPD-S³⁵(inner).
3. When TPD-S³⁵(inner) was given intravenously, urinary excretion of thiamine was delayed compared with that in the case of thiamine-S³⁵ and prolonged retention in the body was noted.
4. With oral administration of TPD-S³⁵(inner), urinary excretion of thiamine was far greater than that in the case of thiamine-S³⁵.
5. Urinary metabolites after administration of TPD-S³⁵(inner) and thiamine-S³⁵ in rabbits were examined and no compounds other than thiamine was detected.

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In order to investigate in detail the increase in blood thiamine level after administration of thiamine propyl disulfide, TPD-S³⁵ (outer), and TPD-S³⁵ (inner), and of thiamine-S³⁵ in rabbits, the distribution of S³⁵ in the blood was examined and the following interesting findings were obtained.
1. The thiamine part of TPD was shown to be found in large quantities in blood cells, while the majority of S³⁵ was found in plasma after oral or parenteral administration of TPD-S³⁵ (outer).
2. S³⁵ was found in the albumin fraction rather than in the globulin fraction of plasma after administration of TPD-S³⁵.
3. After parenteral administration of TPD-S³⁵ (inner), S³⁵ was found mainly in blood cells, and extremely poorly in plasma.
4. The SH group in blood cells is shown to be involved in the mechanism of the penetration of the thiamine part of TPD into blood cells.
5. S³⁵ was increased in blood cells after oral administration of TPD-S³⁵ (inner), though it was less than after subcutaneous injection, whereas the increase in S³⁵ was scarcely noted in blood cells after administration of thiamine-S³⁵. The finding suggests that TPD passes through the intestinal tract without alternation.

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The S³⁵ in various organs was determined after administration of TPD-S³⁵ (inner), TPD-S³⁵ (outer) and thiamine-S³⁵ in the rats and the following results were obtained.
1. Distribution of the S³⁵ of TPD-S³⁵ (inner) and TPD-S³⁵ (outer) differs according to the organ. The former is retained for a longer period of time in the liver and heart and the latter in the blood and intestinal tract.
2. It was found that the replacement ratio of S³⁵ was higher in the heart and brain than in other organs, when TPD-S³⁵ (inner) was given subcutaneously every day consecutively.
3. The accumulation of S³⁵ in the organs was not increased when TPD-S³⁵ (outer) was given subcutaneously every day consecutively, differing from that of TPD-S³⁵ (inner) given in the same way.

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