The potency of the dibenzoylthiamine-hydrolysing enzyme in Takadiastase seems to vary according their lot-numbers. In order to select Takadiastase suitable for dibenzoylthiamine-hydrolysis, a method for the enzyme assay has been set up. From the results of examinations of twenty-six kinds of Takadiastase by this method, it has been confirmed that the Takadiastase which is labelled "For thiamine determination" has the highest potency. It is therefore recommendable for dibenzoylthiamine hydrolysis. Except Takadiastase, no other diastase preparation has been found suitable for dibenzoylthiamine-hydrolysis.

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DAT (Diacetylthiamine) is characteristic in the property of transfering its S-acetyl group to -SH (such as cysteine or glutathione) or -NH_2 (such as aniline) radical whereas it turns into O-acetylthiamine. This transacetylation occurs very easily at the equimolecular basis if the concentration of DAT is high but it proceeds more and more slowly even in excess of cysteine or aniline if the concentration decreases to that comparable to thiamine contents of natural biological materials. Large excess of cysteine is required to complete transacetylation of DAT in its 0.1-1 mg % (0.0025-0.025mM) solution. For example, 0.026 mM DAT solution with 0.064 mM cysteine at pH 7 transfers only 11% of the total S-acetyl group after 24 hrs., while 2.6 mM DAT solution with 3.2 mM cysteine completes transacetylation after 24 hrs. Excess of cysteine such as 2.6 mM is necessary to complete transacetylation of 0.026 mM DAT solution within 24 hrs. The same is true in the transacetylation of DAT by aniline but aniline is less effective to accept the acetyl group of DAT : 2.5 mM DAT solution with 8.6 mM aniline at pH 6 transfers only 15% of the S-acetyl group after 18 days. Human dry plasma (in 2% solution with phosphate buffer) is very strong to convert DAT into O-acetylthiamine ; 100% conversion in 3 hrs. and in the case of 0.2% plasma solution 44-46% conversion in 3 hrs. for 0.0025-0.025 mM DAT solution. Esterase preparation from horse liver hydrolyses DAT into thiamine and in the presence of cysteine hydrolysis of DAT is greatly accelerated.

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Rats were fed for long time on a folic acid deficient diet which was modified from the Ling's deficient diet. The obtained results were as follows : (1) Increase in weight of the rats fed the folic acid deficient diet was remarkably delayed as compared with that of control (fed the pteroylglutamic acid contained diet). (2) The contents of folic acid and citrovorum factor in blood and liver of the rats reared on the folic acid deficient diet were measured by the microbiological assay. Both of them began to decrease after 40-50 days feeding. In 90 day feeding the decrease of them became remarkable and especially, the contents of folic acid and citrovorum factor in liver showed a more marked decrease than those in blood.

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Changes in the contents of folic acid and citrovorum factor in liver and blood were examined by the subcutaneous loading injection of pteroylglutamic acid (1 mg per 100 g body weight) to the rats fed a folic acid deficient diet and to those with the normal diet. (1) The content of folic acid in blood of the two groups showed the highest value in 30 minutes or 1 hour after folic acid loading and returned to normal after 6 hours. (2) The content of folic acid in the liver of the folic acid deficient rats showed 1/3 of the control in 30 minutes after loading. (3) The content of citrovorum factor in blood of the folic acid deficient rats after folic acid loading was lower than that of control, but in 6 hours after loading the normal value was achieved. (4) The content of citrovorum factor in liver of the folic acid deficient rats was lower than that of normal value in 30 minutes after the loading, reaching about half of the control in 1 hour, and after 3 hours it returned to normal.

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Active influenza virus PR8 was inoculated intraperitoneally into two groups of rats, one of them was fed a folic acid deficient diet and the other the normal diet, and the haemagglutinin inhibiting antibody in serum was compared between these two groups. The titre of haemagglutinin-inhibiting antibody in the folic acid deficient group was from 1 : 240 to 1 : 480 and that of control group was 1 : 640 to 1 : 960. It is clear from these results that the deficiency of folic acid brought an inhibitory effect on the production of antibody.

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The method for thiamine determination using Phycomyces blakesleeanus was tested and the use of a Roux flask instead of Erlenmeyer flask was found preferable as it markedly shortens the incubation time and is more suitable for making many assays at a time. A high blank due to cotton plug as has often been observed in microbiological assays was never observed. The pyrimidine and thiazole moieties of thamine have no effect singly on the growth of the fungus, but it showed about the same effect when used in combination.

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A variety of thiamine-derivatives were determined solely or in combination of the pyrimidine or thiazole moiety of thiamine using Phycomyces blakesleeanus Burgeff (-) strain and the relative activities for the fungus were compared. It was found that the fungus is capable of responding to the pyrimidine moiety of thiamine and other various base-exchanged derivatives of thiamine in the presence of excessive amoun of excessive amount of the thiazole moiety of thiamine. For distinguishing the two, a sulfite cleavage method was shown to be useful.

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The reaction products of thiamine by catechol, hydroquinone and rutin, respectively, were isolated in a crystalline form. The three compounds were shown to be identical with thiamine disulfide in respect to ultraviolet and infrared absorption spectra, paper chromatography, elementary analysis and mixed melting point test. The action mechanism of thiaminedecomposing thermostable factor is as follows : Diphenols are oxidized by oxygen to quinones, which in turn oxidize thiamine to thiamine disulfide.

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Previous studies concerning the metabolism of pantothenic acid in patients and animals with liver damage indicated that the deficiency of pantothenic acid frequently exists. (1) Patients : 20mg of calcium pantothenate was administered intramuscularly daily for 1-4 weeks to patients with various liver diseases. After treatment, improvement in various liver function tests was observed. The improvement in hippuric acid synthesis, serum cholesterol ester ratio, and blood pyruvate level were most remarkable. (2) Animals : Characteristic metabolic defects previously described seen in rabbits poisoned with CCl_4,were found to be improved in these animals treated with 10mg of calcium pantothenate daily. These data suggest that pantothenic acid may play an important role in the disturbance of various liver functions.

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Pseudomonas nicotinophaga nov. species was able to grow in a nicotinic acid or nicotinamide medium. The degradation of nicotinic acid or nicotinamide was an adaptive reaction of this organism. 6-Hydroxynicotinic acid was obtained as intermediate product of the degradation of nicotinic acid, while nicotinic acid and 6-hydroxynicotinic acid were obtained as intermediate products of the degradation of nicotinamide. These findings, together with the results obtained from the successive adaptation method by use of the Warburg's manometer, indicate that nicotinamide is first decomposed to nicotinic acid, and nicotinic acid is oxidized to 6-hydroxynicotinic acid.

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This paper presents a determination method for vitamin A esterase activity of animal tissues. Vitamin A esterase activity was recognized in the liver homogenate and pancreas acetone powder incubated at 38℃ for 30 minutes at pH 8.5. A decrease of vitamin A was observed during incubation, but was prevented by the addition of 2 mg of α-tocopherol and 1 mg of ascorbic acid for 100 mg of liver. On determining vitamin A esterase activity, the amount of vitamin A acetate had no effect on the esterase activity when less than 500 I.U. but that of Tween 80,a suface active agent, exerted some effects.

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Vitamin A esterase activity of several tissues and blood of rat was investigated. Optimum pHs of the activity were 8.5,9.0 and 9.7 for the enzymes of the liver and pancreas, for those of the kidney, lung and intestine, and for those of speen and muscle, respectively. Vitamin A-acetate was hydrolyzed with every tissues and blood. Vitamin A-palmitate was hydrolyzed only with the pancreas, but not with any other tissue or blood. The activity of the pancreas and liver was high, and especially that of pancreas highest, but that of the other tissues low. The esterase activity in blood was found to be present not only in erythrocyte but also in leucocyte, but not in the plasma and serum.

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Investigation was made on the influence of hepatic disorder in rats induced with CCl_4 and by the infection with Escherichia coli on the vitamin A esterase activity of rat liver. The result showed that a marked decrease was observed in vitamin A esterase activity of the liver of rat with hepatic disorder experimentally induced.

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Investigations were made on the precipitating reagents for vitamin B_<12>. The results indicated that mercuric ion was no less effective for precipitating dicyanocobalamin than other cations reported such as those of Cu, Co, Ni, Zn, Ag. Further experiments on the precipitating behavior of oxides of the metals described above showed that HgO and Ag_2O had a strong affinity not only to dicyanocobalamin but also to cyanocobalamin, while Fe_2O_3,Co_2O_3,Ni_2O_3,CuO and ZnO had little or no effects on vitamin B_<12> and its dicyanide. The effective metallic oxides tested could be used as a kind of adsorbents for vitamin B_<12>, since they are very difficult soluble in water. Vitamin B_<12> could be recovered very effectively from these precipitates by eluting with aqueous (NH_4)_2HPO_4 solution. Precipitation procedure based on these results would be used for purifying vitamin B_<12> from activated sludge or anaerobic fermentation products of distillers' wastes.

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Investigations were made on the behavior of various solvents to vitamin B_<12> and its dicyanide. The results of the experiments using crystalline vitamin B_<12> indicated that some of lower fatty acids such as n-, iso-valeric acids and n-caproic acid were as effective for extracting vitamin B_<12> as well known phenol or cresol. Vitamin B_<12> extracted with a fatty acid could be transferred to water from the organic phase by simultaneous addition of ether, butyl acetate, ethyl acetate or CaCO_3 with water. Further experiments using aqueous extracts of vitamin B_<12> from natural sources indicated that the solvent extraction procedure using iso-valeric acid followed by butyl acetate was no less effective for purifying B_<12> than that of plenol n-butanol followed by ether reported. Some of these fatty acids seemed to be promising solvents for extracting vitamin B_<12> from viewpoints of the strong affinity and selectivity for vitamin B_<12>, easiness of their recovery and so on.

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