1. Everted sacs of guinea pig intestine were useful to assay the intrinsic factor activity of the purified human gastric intrinsic factor when pernicious anemia patients or totally gastrectomized patients were not available.
2. The author's purified gastric intrinsic factor was found quite active and the activity was destroyed by heating and peptic digestion.
3. Gastric juice from three patients having histamine-fast anacidity without pernicious anemia were confirmed to possess intrinsic factor (IF) by this in vitro assay method.
4. Gastric juice from three patients with non-Addisonian megaloblastic anemia gave greater uptake of ⁵⁷Co-B₁₂ by the sac than that in controls where the sac was incubated with ⁵⁷Co-B₁₂ alone, showing the presence of intrinsic factor in the gastric juice.
5. The gastric juice from pernicious anemia patients showed a rather suppressive effect on ⁵⁷Co-B₁₂ uptake by the sac as compared with the uptake in controls. The presence of the substance inhibiting B₁₂ uptake in the gastric juice of pernicious anemia patients was discussed.

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The purified intrinsic factor (IF) materials obtained from normal human gastric juice by a sequential column chromatography using Sephadex G-100 gel, DEAE-cellulose and Sephadex G-50 gel, were further analysed immunologically for evaluating the purity by immunodiffusion and immunoelectrophoresis techniques combined with autoradiography. The antisera were produced after successive injections of the purified materials with Freund's complete adjuvant. Purification was proved successful by these methods. IF was not detected in the gastric juice from pernicious anemia patients. Other human body fluids and tissues, such as saliva, bile, sera and liver homogenates, were shown to have no IF. Peptic and α-chymotryptic digestions destroyed the B₁₂ binding capacity of human gastric IF, but the effect of tryptic digestion was not marked, when tested by radioimmunodifusion. The significance of this immunological technique was discussed.

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A precise and sensitive method for the fractional determination of thiamine and 2-(1-hydroxyethyl)thiamine (HET) in biological materials was described. Thiamine compounds in tissues were extracted by homogenizing and heating in an acidic medium, and dephosphorylated by phosphatase after deproteinization by metaphosphoric acid. The extract was partially purified by adsorption on permutit and elution with hot KCl-HCl solution. WET in the eluate was determined by a thiochrome method using ferricyanide as the oxidizing agent after destroying thiamine by an alkaline incubation in the presence of a small amount of mercuric ion. Thiamine, on the other hand, was determined without interference of HET by a thiochrome method using mercuric chloride as the oxidizing agent. The reliability of this method was not appreciably affected by the molar ratio of thiamine and HET, and a small amount of HET could be determined precisely in the presence of excess thiamine.

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It was discovered that the reaction of vitamin D₃ with Nield reagent yielded two new compounds, bisteroid A (mp 76-78°) and bisteroid B (mp 98-100°). The molecular formula of bisteroid A was confirmed to be C₅₄H₈₄ and bisteroid B was confirmed to be a monoacetate having the molecular formula C₅₆H₈₈O₂. The chemical structures of bisteroids A and B were studied and assumed structures were given for them.

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The properties of the pyridoxal kinase of mouse brain were studied and the following results were obtained.
1. It was concluded that toxopyrimidine seemed to be phosphorylated by the same phosphokinase as pyridoxal based on the following reasons: the mutual competitive inhibition, the coincidence of the metal requirements, the denaturation effects of heat treatment on the enzyme and the behaviors of inhibitors for both substrates.
2. The substrate specificities of the pyridoxal-kinase were investigated and the following results were obtained: (a) The pyridoxal-kinase was able to act on both certain pyridine and pyrimidine derivatives (b) The 2-methyl of the pyrimidine ring was able to be substituted with C₂H₅ or SCH₃ group for CH₃ group (c) The 4 position of the pyridine ring was able to be substituted with CH₂OH, CH₂NH₂ or CH₃ group for CHO group (d) The compounds without a hydroxymethyl group in the 5 position of the pyridine ring were not phosphorylated at all (such as pyrithioxine) or slightly phosphorylated in such a special case as 5-deoxypyridoxine

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It was found that a new derivative of pyridoxine was formed in the culture filtrate of certain microorganisms which were grown in the medium containing sucrose as a carbon source and pyridoxine. This compound was isolated and characterized its components to be pyridoxine and glucose. The activity of the formation of pyridoxine glucoside was observed in bacteria belonging to the genus Sarcina. Several conditions on pyridoxine glucoside formation by Sarcina lutea were also investigated. Sucrose was the most effective glucosyl donor and maltose was about one-fifth as effective as sucrose, while glucose, fructose, their mixture and other sugars were inactive.

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Various workers have suggested an increased Vitamin B₁₂ requirement in animals fed different high protein diets. From the literature studies, relatively little information has been available on the vitamin B₁₂ status of the animals maintained on high protein rations. An attempt was, therefore, mede to find out the vitamin levels in these animals. Animals were fed two different high protein diets with varied ketogenisity, one having egg albumin as the protein source and the other soybean. Vitamin B₁₂ levels in blood, liver and kidney were found to be depleted considerably in high protein fed animals. Egg albumin which is known to contain more ketogenic amino acids than the soybean protein was found to cause more ketogenesis and more depletion of vitamin B₁₂ in the liver, kidney and blood in the animals. A possible role of high protein diet on vitamin B₁₂ status was discussed in the light of the observations on ketogenic potency of the protein. Glucose cyclo-acetoacetate hydrolysate, which was shown earlier to be a ketolytic compound, was found to prevent, to some extent, the depletion of vitamin B₁₂ levels in high egg albumin fed animals.

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Riboflavin-2′, 3′, 4′, 5′-tetrabutyrate-2-¹⁴C, synthesized from riboflavin-2-¹⁴C, was administered to normal rats per os or by injection. At 72 hours after the administration, the radioactivities of FAD, FMN and riboflavin fractions extracted from the liver, kidney, heart, and small intestine were measured. The results show that the same level of specific radioactivities was found in all flavins of these organs. The specific radioactivities in the case of per os administration were at the same level as those in the case of injection. From these results, it was concluded that the riboflavin moiety of the butyrate administered per os or by injection can be incorporated into FMN and FAD of these organs.

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The definite structure of pyridoxine glucoside which was isolated from the reaction mixture of the intact cells of Sarcina lutea was investigated.
The enzymatic analysis which involved the hydrolysis by yeast α-glucosidase and Aspergillus β-glucosidase, and the oxidation by yeast pyridoxine dehydrogenase were attempted. It was observed that pyridoxine glucoside was slowly hydrolyzed by α-glucosidase and oxidized by pyridoxine dehydrogenase. The spectral change in saturated boric acid solution was also recognized. From these data, the isolated pyridoxine glucoside was presumed to be pyridoxine 5′-α-glucoside involving a small amount of pyridoxine 4′-α-glucoside.
The acetylation of pyridoxine glucoside was available for the isolation of pure preparation of its hexaacetate.
Pyridoxine 5′-α-D-glucoside was chemically synthesized by the condensation of α⁴, 3-O-isopropylidene pyridoxine and 2, 3, 4, 6-tetra-O-benzyl-glucopyranosyl chloride. The synthetic product was identical with naturally occurring one, supporting its presumed structure.

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The formation of pyridoxine glucoside was searched in various kinds of microorganisms which were preserved as type culture and isolated from natural materials. The result of screening showed that the activity was especially found in the bacteria belonging to genus, Sarcina and Micrococcus. The strains of Pseudomonas also produced pyridoxine glucoside-like compound from sucrose and pyridoxine in the culture filtrate.
With the isolated and identified strain, Micrococcus sp. No. 431, several conditions on the formation of pyridoxine glucoside were investigated by growing culture. Sucrose and peptone were the excellent substrate as carbon and nitrogen sources. A few surfaceactive reagents showed the stimulative effect. The maxium values reached 1, 200μg per ml from 1, 000μg of pyridoxine per ml.

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Studies were performed to find out the vitamin B₁₂ status in blood tissues of alloxan diabetic animals. It was found that diabetic animals maintaining higher levels of blood ketone bodies had an increased requirement of vitamin B₁₂. The maximum requirement for the vitamin was found in diabetic animals getting a higher dose of acetoacetate. Hepatic constituents also changed adversely in order of level of ketone bodies in the diabetic animals. Some direct or indirect interrelationship between vitamin B₁₂ with ketone bodies are suggested.

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The specific calcium-binding factor was fractionated from heat-treated rat intestinal mucosa supernatants with use of ⁴⁵Ca-equilibrated Sephadex G-25.
Calcium-binding fraction was calcium specific, and consisted of two components. The first component was increased by vitamin D₃ administration, suppressed with actinomycin D pretreatment, and decreased by trypsin digestion. On the other hand, the second component was not influenced by vitamin D₃, but increased by trypsin digestion.
From these results, this factor would be protein. The second component might be a smaller fragment of the first component. Vitamin D₃ would stimulate directly or indirectly the synthesis of specific calcium-binding protein.

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