In the previous paper the author reported riboflavin is extremely rich in the kidney of marlins. Murayama and Randoin have found abundance of riboflavin in the kidney of pink salmon and beef, amounting 11.8 and 10.0mg per cent on fresh basis, respectively. The author's data on marlin kidney, however, showed far richer in riboflavin amounting thirty times, at the maximum, as much as the above animals. Analytical test, paper chromatography, absorption spectra and fluorescent spectra have confirmed that the so-called riboflavin in the marlin kidney to be identical with the true riboflavin.

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In the previous paper it was shown that the decomposition of FAD was greatly accelerated in the presence of trace amounts of heavy metal ions which were brought into the solution from the glass ampule, and that this decomposition was prevented by adding a small amount of EDTA. In this paper, examinations were made on the stabilization of FAD in aqueous solution using EDTA and its analogues, such as polyphosphate, oxine derivative, dicarboxylic acid, hydroxycarboxylic acid and natural amino acids as the heavy metal sequestering agent. Among these compounds, EDTA, 1,2-cyclohexanediaminetetraacetic acid, diethylenetriaminepentaacatic acid or hydroxyethylenediaminetriacetic acid, had a better stabilizing effect on FAD solution. This effect was also observed with sodium hexamethaphosphate, sodium pyrophosphate, 8-hydroxyquinoline-5-sulfonic acid, citric acid, malonic acid, malic acid, tartaric acid or nitrirotriacetic acid but not with amino acids. It is concluded that the larger the chelate formation constant of sequestering agents is, the superior its stabilizing effect on FAD solution is.

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S-Benzoylthiamine monophosphate and S-benzoylthiamine were detected on filter paper by thiochrome reaction after reduction with cysteine into thiochrome positive form. In contrast to aqueous solution, the thiochrome formation on the paper was strongly hindered by the presence of cysteine, thus giving only very weak fluorescence. However, the increase of the intensity of the fluorescence was observed by spraying of mercuric chloride before the formation of thiochrome, as excess SH group of cysteine was oxidized. The procedure was as follows : after spraying of 1% cysteine solution (pH 6.5) and heating for 30 minutes in a fume chamber, the paper was sprayed with 5% HgCl_2 and finally with a mixture of 2 parts of 96% ethanol, 1 part of 10% NaOH and 0.05 parts of 2.5% K_3Fe (CN)_6. This method would be applicable for detection on filter paper of other thiamine derivatives with open thiazole ring.

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A modification was made of the components of basal medium for the determination of thiamine with Lactobacillus viridescens. In place of tryptone and thiamine-free yeast extracts in APT medium reported by Deibel et al., commercially available polypepton and yeast extract were used after having been treated at 15lb for 25 minutes in an autoclave in the presence of an appropriate amount of sodium hydroxide, and then neutralized. It has been found that the assay method using the modified medium is as specific, reproducible, and convenient as that using APT medium. Comparative investigations of the activities of a variety of thiamine derivatives on L. viridescens were made under different conditions. Thiamine propyldisulfide, thiamine dicetylsulfate and thiamine diphosphate were as much active as, and S-benzoylthiamine monophosphate was less active than thiamine hydrochloride. S-Ben-zoylthiamine and O, S-dibenzoylthiamine showed a slight activity when autoclaved with the basal medium. A similar behavior was observed when thiamine derivatives dissolved in sterile water were added to the sterile modified medium. The activity of S-benzoylthiamine monophosphate seems to be higher in the modified medium than in APT broth. S-Benzoylthiamine and S-benzoylthiamine monophosphate were more active when autoclaved with the medium containing cysteine than without the substance.

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The authors isolated TAO (thiamine anhydride sulfoxide) (I) and 2-methyl-4-amino-5-aminomethylpyrimidine (II) from the urine after intraperitoneal injection of TA (thiamine anhydride) to rats on thiamine-deficient diet. The evaporated urine was extracted by n-butanol, The extract was purified by chromatography using Amberlite IRC-50 and finally from the eluate an alcohol-insoluble crystal of mp. 260℃ and an alcohol-soluble crystal of mp. 130℃ were isolated. The former was identified as II and the latter coincided with I. As both TAO and thiamine anhydride sulfone show the same mp. and Rf on paper chromatography, the metabolite of mp, 130℃ was identified as I by polarography which will be described in the next paper. It is questionable whether II was a real metabolite of TA, because at first no spot coincided with II was not detected by paper chromatography of the original urine and TAO is readily hydrolysed into II by its acid treatment.

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Oxidation of thiamine anhydride by hydrogen peroxide resulted in its sulfoxide (I) or sulfone (II) according to the reaction media. The authentic specimens of I and II were prepared, and checked both by elementary analysis and by infra-red spectrography. Both I and II show the same mp. of 132℃ and the mixture of I and II also indicates the same melting point. But after drying the samples at 60℃ for more than 60 hours, the both specimens were elevated to mp. of 177℃ (I) and of 155℃ (II). Furthermore, PbO_2-oxidation of TA, I and II revealed different degrees of PbO_2 consumption which were measured by polarography. TA, I and II each consumed about 3,2 and 1 atoms of oxygen after one hour of the reaction, The metabolite isolated from the urine of rats after intraperitoneal injection of TA also indicated the same picture of PbO_2-consumption as I. Therefore the metabolite which coincided with I as shown in the previous paper, was proved as I.

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Some examinations were made in order to know the native forms of cobamides synthesized in Pr. shermanii cells grown in culture medium containing X-factor and 5,6-dimethyl-benzimidazole (DBI). The bacterial cells were harvested by centrifugation in the cold and homogenized in a glass-homogenizer with absolute alcohol. The homogenate was centrifuged and the supernatant was diluted to an appropriate concentration and then applied to Toyo No.50 paper. Electrophoresis at 13V/cm in 0.5N acetic acid was performed for 3 hours. For detecting E. coli-active substances on paper, bioautographic technique on agar-plate was employed. The results obtained indicated that at least 4 kinds of cobamides existed in the cell-extract. Of them, one that moved most rapidly towards cathode was seemed to be Barker's DBCC, second one that moved towards anode would be protein-bound cobamide. Other two substrances were similar to cyanocobalamin and cobinamide in respect of electrophoretic behavior.

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The tube assay methods of lipoic acid with Streptococcus faecalis R by Yamasaki et al. and with Corynebacterium bovis 187 by Stokstad were applied to the bioassay of lipoic acid in human urine. The data of same urine samples obtained by the two methods were almost equal, but the recovery of added DL-lipoic acid was not satisfactory in the method with C. bovis, though that of the method with Stc. faecalis R was enough. It is supposed that the growth response of C. bovis for lipoic acid in urine might be inhibited by some substances in urine. The biological activities of some lipoic conjugates for the both microbes were compared. N-DL-lipoyl-DL-glutamate was found to be fully active for the growth of C. bovis, while almost inactive for that of Stc. faecalis.

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A tube assay method with Streptococcus faecalis R for the determination of total lipoic acid in urine was investigated. The urine samples were hydrolyzed by autoclaving for 1 hour with 6N HCl under nitrogen and in the presence of about 5mg per tube of bovine serum albumin. The hydrolyzed mixture was diluted, neutralized and the aliquots were allowed to the determination by the tube assay method. The recoveries of lipoic acid added to the urine were complete and the estimated values of lipoic acid were highest among other treated samples. Conjugated lipoic acid in urine were not hydrolyzed with some proteolytic enzymes or lipoamidases.

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A chemical determination mothod for lipoic acid was investigated according to Hager who applied the method to the study of dihydrolipoic dehydrogenase. It was based on the reaction that sulfhydryl groups reduce ferricyanide quantitatively to ferrocyanide and the ferrocyanide formed is measured colorimetrically. The conversion of lipoic acid in samples to dihydrolipoic acid was done with 10mg of NaBH_4 and 0.3ml of 10% HCl was used in order to remove the excess of NaBH_4. Optimum amounts of lipoic acid to be determined were 5 to 50mμg in samples. This method can be applied to the determination of pure lipoic acid preparation, but it was not adequate to that of the small amount of lipoic acid in biological samples.

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In investigation of the biological activities of some lipoic acid derivative for the growth of Streptococcus faecalis R and Corynebacterium bovis 187,it was found that lipoylglutamate was active for Corynebacterium bovis alone. To clarify this mechanism, the enzymatic activies of extracts obtained from both microbes on lipoylglutamate was studied. Corynebacterium bovis contained an enzyme which liberates lipoic acid from lipoylglutamate, on the other hand no activity of the extracts from Streptococcus faecalis R was found. The enzyme was partially purified and the some properties were demonstrated. Optimal pH 6.0,optimal temperature 37℃ and the enzyme reaction was inhibited with some heavy metals and SH-inhibitors.

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Investigations were made on the absorption and excretion of S-carbalkoxythiamine (CAT). When CAT was administered on healthy man per os, the thiamine level of blood and thiamine elimination in urine were higher than in case of thiamine hydrochloride administration. This fact shows CAT is easily absorbable than thiamine hydrochloride. In the case of injection into the gut of dog or rat, CAT was found in itself in portal vein, but not in liver. Thus it is supposed that CAT is absorbed from gut in itself and a part of it is hydrolysed to thiamine in portal vein and the remainder in liver.

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