Urinary excretion of formiminoglutamic acid (FIGLU) and urocanic acid after a loading with 15g of L-histidine was observed in 53 patients with various liver diseases. Of 53 patients 58.5% was in excess of FIGLU, 52.0% for urocanic acid, and 64.0% for the two combined derivatives. The frequent abnormality in these histidine derivatives in liver diseases suggested a widespread deficiency in folic acid metabolism in such patients.
For analyzing the mechanism of an excessive excretion of histidine derivatives, the therapeutic trials with minimal daily doses of folic acid, tetrahycro-folic acid (THF), and doses of coenzyme B₁₂ and CN-B₁₂ were carried out in seven patients with liver diseases and changes in the urinary histidine derivatives were observed.
Four out of seven patients responded to folic acid, and oneof two patients refractory to folic acid responded to THF.
It was of particular interest that a case in which both folic acid and THF were ineffective, responded to both coenzyme B₁₂ and vitamin B₁₂. The mechanism of an increased excretion of histidine derivatives in the liver disease was discussed.

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Hog intrinsic factor (IF) was purified by DEAE-cellulose batchwise elution and DEAE- and CM-cellulose column chromatography. Only one B₁₂ binding protein (PHIF) was obtained, which was IF-active at the dose as low as 0.4mg. PHIF had B₁₂ binding capacity of 23.0μg/mg protein when studied by dialysis, and was found homogeneous, when studied by paper electrophoresis, ultracentrifugation and absorption spectrometry. The molecular weight of PHIF was calculated from the sedimentation coefficient of 3.4S, and was found to be about 50, 000.
On chemical analysis, it consisted of 18 amino acids, and hexose, hexosamines, fucose, and sialic acid. The composition of amino acids was determined.
The possible model of B₁₂ binding was also discussed on the basis of the molar ratio of PHIF's molecular weight to B₁₂.

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Using Ouchterlony's agar gel double diffusion and radioautography, the physicochemical properties of hog intrinsic factor and the probabilities of existence of hog intrinsic factor-like substance in various hog organs, such as liver, spleen, bile, serum, etc. were investigated.
1. Hog intrinsic factor (IF) has resistance to 8 M urea and papain treatment in a bound or unbound form, and to 45% acetone and to pepsin treatment, if it was bound to B₁₂.
2. Hog IF was a little resistant to Nagase treatment, and heating to 100° within 30 minutes. It was completely destroyed by HCI treatment for 24 hours, pepsin treatment in a unbound form, α-chymotrypsin and trypsin in a relatively high concentration, 45% ethyl alcohol in both forms, and 45% acetone in an unbound form.
3. By immunoautoradiography hog IF-like substance was found in the hog bile, serum, spleen, liver, but not in the kidney and thyroid gland. It suggests that hog IF-like substance might be contained in these organs.

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One gram of raw hog intrinsic factor (IF) was administered to each of ten control subjects with no hematological disorders, three pernicious anemia patients, and three total gastrectomy subjects. A B₁₂-binding substance originating from raw hog IF was detected in the sera and urines of seven control subjects, after the oral administration of hog IF, and the serum level of the binding substance was assumed to be 5.0μg/ml or slightly more in the detected cases. In the remaining three control subjects and all cases with pernicious anemia or with total gastrectomy, the substance was not found in their sera and urines either before or after the oral administration of raw hog IF. Based on the finding that the detected substance was immunologically identified with the purified hog IF, it was concluded that “immunoreactive hog IF” or rather hog IF or its split fragment was absorbed from human intestine. The significance of the negative demonstration of immunoreactive hog IF in pernicious anemia and in total gastrectomy was also discussed.

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The enzyme destorying FAD in spinach leaves were investigated. 1. FAD was hydrolyzed by nucleotide pyrophosphatase in spinach leaves. 2. The activity of the enzyme was considerably high in comparison with that of FAD pyrophosphorylase in green leaves. 3. The pH optimum was around 5.0 and the activity decreased remarkably at about pH 7.0, which was the pH optimum of FAD pyrophosphorylase. 4. The optimum temperature for the enzyme activity was approximately 60° when the reaction mixture was incubated for 20 min. 5. The Km value was 4×10^-5M and the relative substrate specificity of the enzyme for FAD was higher than those for NAD, NADP and ADP. 6. The hydrolysis of FAD was strongly inhibited by ATP, ADP, NAD and NADP occurring in green leaves about 10 times as much as flavins in concentration. 7. No hydrolysis of the tightly bound FAD with the apoprotein of glucose oxidase was recognized.

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Properties of a system of thiamine propyl disulfide (TPD) uptake have been investigated in Escherichia coli. It was found that the cells incubated with TPD in the presence of glucose at 37° accumulated thiamine pyrophosphate in the cytoplasm with a negligible amount of TPD. TPD in the reaction medium remained practically in the unchanged form after incubation with the cells for 30 minutes at 37°. Thiamine pyrophosphate accumulation in TPD uptake was an energy- and temperature dependent process which follows Michaelis-Menten kinetics; apparent Km was 3.0×10^-6 M and Vmax was 3.5×10^-5 moles per minute per g dry weight of the cells.
Treatment of the cells with N-ethylmaleimide resulted in inhibition of the rate of thiamine pyrophosphate accumulation much stronger in TPD uptake than that in thiamine uptake. A thiamine transport-negative mutant of E. coli which is defective in the “carrier” protein specific for thiamine, did not accumulate thiamine pyrophosphate when incubated with TPD.
These results strongly suggest that in E. coli TPD is not directly taken up by the cells but is first reduced to thiamine on the outer surface of the cell membrane probably by a non-enzymatic process. Thiamine thus formed is transported via the uptake system specific for thiamine; the “carrier” protein functions for passage through the membrane and membrane thiamine kinase functions to accumulate thiamine as thiamine pyrophosphate in the cytoplasm.

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1. The action of thiamine tetrahydrofurfuryl disulfide (TTFD) on the excised heart of clam (Meretirx meretrix lusoria) was investigated.
2. TTFD displayed a striking postive inotropic action. This action did not appear in Ca-free or Na-free solution, but appeared in K-free solution.
3. TTFD antagonized the negative inotropic action of acetylcholine.
4. These actions of TTFD were closely similar to those of thiamine propyl disulfide and these actions may be ascribed to -N-CHO -C-CH₃=C in the structure.

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The significant diaphorase activities which could not be explained by contaminating mitochondria and microsomes are consistently present in isolated calf thymus nuclei. The nuclei seemed to contain at least two apparently different diaphorase activities. Most of the NADH-ferricyanide reductase activity was retained in the nuclei purified by sucrose-gradient centrifugation and sonication. Triton treatment which remove cytoplasmic contaminants and the outer layer of nuclear membrane resulted in no decrease of NADH-ferricyanide reductase activity in nuclear preparation.

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The separation method and some properties of isodynamic acid phosphatases catalyzing FMN hydrolysis in spinach leaves were investigated.
1. The enzyme catalyzing FMN hydrolysis was found to occur practically and exclusively in the final supernatant fraction by differential centrifugation.
2. Acid phosphatase catalyzing FMN hydrolysis was separated into three fractions by chromatography on Sephadex G-100, equilibrated with 0.2M glycine buffer (pH 9.0).
3. pH optima of the three fractions were similarly 5.0.
4. Each affinity of the three enzymes for FMN (Km value) were calculated to be 1.8×10^-4 (Fraction I), 1.2×10^-4 (Fraction II) and 7.0×10^-4M (Fration III), respectively.
5. The substrate specificity for the three acid phosphatases was tested, and it was found that Fraction III was comparatively specific for FMN.
6. Fractions I and II were inhibited by ATP, ADP, orthophosphate and pyrophosphate, whereas Fraction III was inhibited by AMP and adenosine.

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The effects of vitamin D₃ and actinomycin D on the radioactive calcium distribution were studied in the rat intestinal mucosa and muscle 5 and 30 minutes after the oral administration of ⁴⁵Ca.
1. Serum ⁴⁵Ca concentration at 30 min was increased significantly in the vitamin D₃ treated rat, and this vitamin D₃ effect was inhibited by actinomycin D. The action of vitamin D₃ and actinomycin D was not observed at 5 min. 2. ⁴⁵Ca was observed in the proximal parts of intestinal mucosa (from pyloric end to 30cm) at 5 min, and at 30 min it came to be observed in the distal (40-60cm). ⁴⁵Ca in the proximal parts at 5 min was reduced with vitamin D₃, and actinomycin D did not seem to be influenced on this vitamin D₃ action. ⁴⁵Ca in the distal (at 30 min) was enhanced with vitamin D₃ and this action was partially inhibited by actinomycin D. 3. ⁴⁵Ca in muscle was lower than in mucosa. Observed pattern of ⁴⁵Ca distribution at 5 min in muscle was not so different in vitamin D deficient and D₃ treated rats. Actinomycin D did not exert its action on muscle at 5 min. Distribution patterns of ⁴⁵Ca in muscle at 30 min were almost similar to the mucosal patterns. 4. Subcellular distribution of ⁴⁵Ca was observed in the duodenal mucosa. Nuclei and cell debris (brush borders) have relative amounts of ⁴⁵Ca 5 min after ⁴⁵Ca administration. At 30 minutes, ⁴⁵Ca of subcellular fraction was significantly decreased, especially in nuclei, cell debris and mitochondria. This reduction was moderated by vitamin D₃. Actinomycin D inhibited this vitamin D₃ action in microsomes and supernatants.
From these results, it was suggested that calcium transport would be carried out in two ways, membrane permeable system (actinomycin D insensitive) and the system involving calcium carrier protein (actinomycin D sensitive).

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The nutritional requirement of L. bulgaricus B-1 strain have been studied. By the culture of the strain in a modified Thompson's medium, it was found that the strain possessed very complex requirements of growth factors. The strain required L-tyrosine, L-cystine, nicotinic acid, riboflavin, enzymatic digest of casein, and Tween 80 in addition to pantethine and pantothenate.
Elongation of the strain was found in the absence of vitamin B₁₂ and folic acid, but it could be prevented by addition of vitamin B₁₂ and folic acid.
It was of interest that the mixture of the enzymatic digest of casein and Tween 80 became toxic to the strain at higher concentrations, though each substance stimulated the growth of the strain in an amount up to 100 and 50mg per 100ml of the basal medium, respectively. The strain was also found to be useful for the microbiological assay of nicotinic acid, nicotinamide, enzymatic digest of casein, and Tween 80 in addition to pantethine.

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A microbiological assay procedure for pantethine in the absence of pantothenic acid has been presented, based upon the growth response of L. bulgaricus B-1 strain.
1. The optimum concentrations of Tween 80 and enzymatic digest of casein for the growth of the strain were 50mg per 100ml of the basal medium.
2. The addition of L-glutamic acid and L-tyrosine promoted the growth rate of the strain and the addition of L-tyrosine could especially reduce the incubation time from 40 to 20 hours.
3. From the above result an appropriate basal medium as described in Table 2 was devised.
4. The recovery of Pantethine added to two pharmaceutical preparations was 95-98 per cent when tested by the devised method.

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Induction of fatty liver in rats by feeding on diets containing 1.5% orotic acid for 7 days, was accompanied with the increase in the lipid TBA value and with the decrease in the lipid phosphorus concentration. However, fat accumulation was prevented by iniecting 30mg of myoinositol or choline subcutaneously every day during the experimental period. The prevention was found to be about 20-50% with myoinositol and about 90% with choline. However, the effects of choline and myoinositol were not synergistic; the effect due to the injection of both choline and myoinositol was rather close to the effect of myoinositol alone. The increase in lipid TBA value and the decrease in lipid phosphorus concentration were also found to be prevented by injecting these lipotropic factors.

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