The relative potency of thiothiamine to thiamine was again assayed by prophylactic test with rats and mice and by Lactobacillus fermenti assay and it was found to be about 1/100,1/50 and 1/20000,respectively. Thiothiamine showed no thiamine effect both in rice bird assay (0.4μM) and in rat bradycardia test (1.0μM). These results led to the conclution that thiothiamine had almost no thiamine activity and confirmed the findings in rat growth assay by Suhara et al. Discrepancy between the results of the previous and present reports was also discussed.

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The difficult soluble thiamine derivative, such as dibenzoyl thiamine, thiamine dicetyl sulfate or thiamine naphthalene-2,6-disulfonate was dissolved in hot water and sprayed on polished barley before the pressing process. The enriched barley, thus prepared, retains the vitamin without the considerable loss by washing.

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The relative weights, histological changes, cholesterol contents and reduced glutathione contents of several organs of the control and pantothenic acid deficient rats were examined for several weeks experimental period. In addition, the lymphocyte count in blood, glucose-tolerance-test and acetylation power of the control and pantothenic acid deficinet rats were examined. And, finally, relations between some symptoms of deficient rat and its adrenal function were studied by adrenectomized rat of pantothenic acid deficiency. These investigations indicated that after a few weeks on a deficient diet, the secretions or the activity of cortisone-like substance increased and stage of adrenal insufficiency became subsequently.

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The author studied the correlation between estrogen and vitamin A in albino rats. An administration of estrogen causes a reduction of vitamin A content in the body. A large dose of vitamin A shows an antagonistic action against estrogen, while the function of the liver concerning inactivation of estrogen is not affected by vitamin A

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Investigations were made on the change of absorption spectra of vitamin A and its related compounds, derived from β-ionone by the addition of antimony trichloride or boron fluoride ethyl ether. Changes of absorption spectrum as well as transfer of absorption maximum were observed in some compounds, and hyperchromic effect in other compounds. Similar results were obtained in the compounds with benzene ring.

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No significant increase of urinary riboflavin could not be recognized in the normal children given orally 0.2 mg per kg of body weight of TPD for ten days. In case of oral administration of 0.4 mg per kg of TPD through catheter for 28 days to healthy infant under artificial feeding, the urinary excretion of riboflavin seemed to increase in the first week after the administration, but it tended to decrease in the second week. On the other hand, a trasient decrease of riboflavin was recognized in the blood of bottle-fed infant similarly treated for 21 days. Generally, the administration of TPD caused a temporary increase of FAD in the blood soon after the treatment but a gradual decrease thereafter, and an increase again after three weeks. FMN in the blood was in an inverse relation with FAD against the administration of TPD, while a decrease of free riboflavin was observed soon after the treatment. Neither normal children nor the healthy infants under artificial feeding showed any clinical symptoms of riboflavin deficiency such as glossitis, angular stomatitis, and cheilitis.

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Parenteral administration of 4 mg per kg of body weight of TPD to healthy bottle-fed infant caused by effect three days after the administration. In case of 2.0 mg per kg, an increase of urinary riboflavin was observed, especially in that six hours after the administration. Chromatographic investigation indicated that the increase was due to mainly to free riboflavin. The administration of riboflavin esters did not cause the increase in urinary FAD or FMN.

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Investigations on the chromatographic developing agents for riboflavin and its derivatives showed that a mixture of n-butanol・acetic acid・water (4 : 1 : 5) was suitable for separation of the vitamins, and the use of an aqueous solution of 5% Na_2HPO_4 enabled the elimination of interfering substances for the estimation of riboflavin in the urine since the Rf value of the substances became larger than those of the vitamins. For paper chromatographic determination of three forms of riboflavin, it is necessary to employ two dimensional procedure using n-butanol・acetic acid・water as a primary solvent and 5% Na_2HPO_4 as a secondary one. High riboflavin concentration and low interfering fluorescence in the urine of healthy infants fed with riboflavin-enriched powdered milk enabled the separation from interfering substances and estimation of riboflavin, while in case of normal children without supplement of riboflavin the fluorometric estimation of the vitamin was difficult due to the low vitamin as well as high interfering fluorescence. No yellow fluorescent band corresponding to FAD could not be recognized in the present experiment.

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Investigations were made on microbiological assay of thiaminase with Lactobacillus fermenti 36 ATCC. Qualitative test for thiaminase was possible using the growth inhibition of the bacillus after preliminary diffusion of the enzyme. In case of Bacillus thiaminolyticus Matsukawa et Misawa an antibiotic substance was found to be produced in the medium but its effect was negligible when the diluted medium was used for the test. Quantitative assay of thiaminase by Lactobacillus fermenti was compared with the chemical method, and it was found to give apporoximately similar results.

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The relationship between the growth of bacteria and the produdtion of thiaminase was studied by culturing Clostridium thiaminolyticum anaerobically with various methods. In peptone water, it grew best with the double tube method, in which least oxygen was dissolved. In neutrient broth, it grew well in any method used, but the less oxygen was dissolved, the more thiaminase was produced. Thus the growth of the bacteria and the production of thiaminase was found to be dependent on the condition of culture.

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Thiamine-degrading bacteria have been isolated from canine feces collected in Osaka Prefecture. Five strains of Bacillus thiaminolyticus and fifteen strains of Clostridium thiaminolyticum were isolated from 87 canines. Comparative studies were made for isolating thiamine-degrading bacteria with both microbiological assay and chemical method and the usefulness of micrbiological method was ascertained.

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Thiamine anhydride (TA) was isolated from the alkaline solution of thiamine with benzenesulfonyl chloride (BSC) after its overnight standing at room temperature but thiamine disulfide(TDS) (I) and its diphenylsulfonate (II), instead of TA, were obtained by the same procedure under ice-cooling. Bessey's method of estimating thiamine by alkaline BSC-treatment also depends on the same inactivation of thiamine. We checked whether formation of TDS or its disulfonate is possible during the estimation procedure by the following ways : (1) Thiamine in 0.1〜l000mg% solutions was inactivated by addition of BSC and N NaOH completely ; no thiamine was detected by thiochrome method. After the inactivation, TDS was transformed into thiamine by means of cysteine-treatment at pH 5.5 and determined by cyanogen bromide oxidation ; 2,1,5,5 and 9% of the Initial thiamine contents were found as TDS respectively to 0.1,1,10,100 and 1000 mg % concentration of thiamine. If the inactivation by BSC was carried out under ice-cooling, formation of TDS was increased to 3,5.5,9,12 and 16 % at the corresponding concentrations. (2) Thiamine-phenyl-sulfonate (III) was converted into TA easily and completely by making its solution more alkaline than pH 10 ; no TDS was proved by means of cysteine treatment. On the other hand TDS diphenylsulfonate was transformed into TA slowly and incompletely by making its solution even more alkaline than pH 12. Thus it was proved that thiamine was transformed into TA throgh thiamine-phenylsulfonate (thiol-form) (IV) by the alkaline BSC-inactivation and formation of TDS was less than 2% at dilute concentration such as used in the estimation procedure.

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The improvement of vitamin B_<12> productivity of Streptomyces olivaceus by induced mutation was made with ultra-violet light as a mutagenic agent. The mutant with the productivity of from 2.2 to 2.3 μg of vitamin B_<12> per ml was obtained after four generations, but further improvement was unsuccessful after ten generations under our experimental conditions. All the mutants obtained had the same morphological characteristics as in the original culture except for one albino and two yeast types. Only one culture lacking vitamin B_<12> productivity was obtained.

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In our previous papers, it was shown that the bacterial cell fraction obtained by centrifugation from supernatant liquor of methane fermentation digester of distillers' wastes contained high vitamin B_<12> activities and might be useful as an industrial raw material for the preparation of crystalline vitamin B_<12>. Therefore, in order to collect the new vitamin B_<12> resources, the Sharples type supercentrifuge was operated 20 times at the Ishioka Distillers' Factory in Ibaraki Prefecture. About 16 kg of the dried cell fraction (13.5% moisture) was obtained, corresponding to 0.4% of the supernatant liquor treated. The vitamin B_<12> content of the mixed sample of bacterial cell fraction, estimated with E. coli, L. leichmannii and Ochromonas malhamensis, was 50,43 and 30 μg/g on the dry basis, respectively. The supernatant liquor of distillers' wastes digester seems therefore to be superior to that of night soil digester in view point of high yield and high vitamin B_<12> content.

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Fundamental experiments on column partition chromatography for vitamin B_<12> were conducted with the view of selecting the solvent system and the innert supporter most suitable for applying the technique to one stage of processes for manufacturing crystalline vitamin B_<12> from activated sludge. In the first place, various solvent systems which contained n-butanol as a main component were examined, using powdered cellulose column. In case of water-saturated n-butanol, R value of the vitamin was about 0.24 and the degree of diffusion of zone was moderate. The addition of HCl, NaCl, KCN or phosphate to the system resulted in larger R value, but exerted little influence on the degree of diffusion. Also, organic compounds, such as methanol, iso-propanol and phenol, were used as an additional component in solvent systems and proved to promote the rate of movement of zone. Secondly, tests were made on the systems composed of sec-butanol and water in different proportions. Water-saturated sec-butanol was unsuitable, because it caused a considerable diffused zone. However, more distinct zones were obtained when water added to sec-butanol was reduced. Finally, our comparison tests on several kinds of innert supporters showed that powdered cellulose was most suited.

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Investigations were made on the availability of column partition chromatographic procedure for one of the processes for the manufacture of crystalline vitamin B_<12> from activated sludge. Partially purified vitamin B_<12> solutions having purities of 0.5〜0.9% were used as sample solutions. By developing with solvent systems containing n-butanol as a main component and by collecting the appropriate fractions, vitamin B_<12> solutions of 7〜42% could be obtained. It is concluded from these results that the column partition chromatographic method could replace the ion exchange chromatographic procedure which had been adopted in the previous pilot plan. The evidence was also supplied suggesting the presence of a compound closely related to cyanocobalamin in the extract from activated sludge.

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Further investigations on column partition chromatographic purification of crude vitamin B_<12> solutions were pursued using aqueous solutions with lower purities than those used in the previous report, since it was presumed that the purification efficiency by the method would be dependent on the purity of the original solution. The solution used was prepared by eluting the vitamin from charcoal adsorbate with 60% aqueous acetone and by concentrating eluate under reduced pressure. Of three kinds of solvent systems examined, n-butanol saturated with phosphate buffer solution of pH 8 was found to be most efficient. In this case, the purity of the solution obtained by collecting appropriately the fractions from the column was 3.3%, and it corresponded to about 50 times of that of the original sample solution. This result is superior to our results reported previously. In addition, influence of amount as well as concentration of solution to be applied onto column upon the purification efficiency was studied.

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Several conditions were investigated for the extraction of vitamin B_<12> from the dried bacterial cell fraction obtained by centrifuging supernatant liquor of night soil treatment plant. Extraction of the vitamin was satisfactory by boiling for 30 minutes with 10 parts of 0.01〜0.1% KCN aqueous solution added to the raw material. Unless cyanide was present, vitamin B_<12> activity of extract proved low, especially with the Ochromonas assay.

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The present paper concerns with further investigations on the extraction of vitamin B_<12>. The object was the dried cell fraction obtained by centrifuging the supernatant liquor of distillers' wastes digester. The results were similar to those in our previous paper. However, the values for vitamin B_<12> activity of the extracts obtained with aqueous solution of S-containing compounds, such as Na_2S_2O_5 and Na_2S_2O_4,were comparable to the activity of the extract with cyanide solution, when E. coli or L. leichmannii was used as a test microorganism. The use of cyanide may thus possibly be avoided.

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Choline content in the blood was determined in various diseases and its change by medical treatments was observed. The average choline content in the blood of 38 healthy subjects was 20.2±1.25mg/dl, and no differences were found between male and female, or young and old. The choline content in the blood was high in bronchial asthma, jaundice, chronic glomerulonephritis and leucemia, while it was low in tuberculous diseases, sepsis, gastritis, gastroduodenal ulcer, splanchnoptosis, colitis, dysentery, cirrhosis of liver, neuropathy, anemia, endocrinism, avitaminosis and acute toxicosis. It was found that the determination of choline content in the blood was useful for the judgement of hepatic and posthepatic jaundice, and in the latter the choline content was over 25 mg/dl in every case. The administration of thiamine, riboflavin, nicotinic acid or insuline, blood trasfusion or mixing of blood in vitro, caused a decrease of choline content in the blood while the administration of multivitamin preparation, ATP or lipotropic factor or X-ray irradiation caused its increase.

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Investigations were made on the urinary excretion and transfer to milk of riboflavin parenterally administered as well as its concentration in the blood prior to and after the administration of riboflavin to women who progressed on a normal course of pregnancy and labor. The requirement of mothers for riboflavin increased at the end of pregnancy, but it decreased during the period from parturition to the beginning of lactation, while the requirement of mothers nursing sufficiently was larger than that of insufficient lactation. Subcutaneous injection of riboflavin indicated that the rate of increase of the vitamin in the milk was highest 2 and 3 hours after its administration. No remarkable change was recognized in the riboflavin concentration in the blood before and after the delivery.

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Anterior pituitary hormone (100 I.U.) was daily injected into rabbits for 4 weeks, accompanied by a loading with 1 mg of riboflavin weekly during the period of the administration. In this experiment, the changes of both the riboflavin concentration in the blood and its urinary excretion were pursued on the basis of time-unit. In every case, similar tendencies were observed in the blood riboflavin levels prior to and after the administration of anterior pituitary hormone, while in the urinary excretion of riboflavin for 4 hours after the riboflavin injection, no significant difference was not recognized between pre- and post-administration of the hormone.

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