To determine the effects of oral administration of homopantothenic acid (HOPA) on electroencephalogram of mentally retarded children for a long term and to elucidate the duration of effect of this substance after withdrawal of the drug, we studied the EEG of these children by means of automatic frequency analysis. In the cases given 50 mg/kg of HOPA continuously for two years, its effect on the basic pattern was most marked by one month after starting the administration and its effect persisted thereafter. When 50 mg/kg of HOPA was administered for one year and changed to placebo for another year, EEG was found to revert to the pre-treatment conditions 3 months after withdrawal of the drug. In the determination of effective minimal dose of HOPA by frequency analysis of EEG, 25〜30 mg/kg proved to be ineffective and 40〜50 mg/kg was supposed to be critical dose. When HOPA is given to patients with clinical epilepsy, the frequency of seizures was generally aggravated, and this tendency became more marked in children with focal cortical seizures.

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In order to investigate the influence of homopantothenic acid (HOPA) on the ultrastructure of liver cells of young rats, fed on pantothenic acid (PaA)-deficient diet, was administered with HOPA at a daily dose of 1g/kg for 4 days (group 1) and compaired with rats supplemented by PaA (group 2). In group 1,the hepatic cells were atrophied and appeared to be somewhat dark, as "shrivelled necrosis". The cytoplasmic organelles were increased in number and looked compact lacking the cytoplasmic matrix. The mitochondrion was increased in number and size, and showed a curious deformity. The outer membrane showed a toothed-wheel structure and the cristae appeared a wheel-axle pattern. The microbody was increased in number with bizarre figure. The crystalloid in the matrix was disappeared. Rough surfaced endoplasmic reticulum (RER) was almost disappeared and the cisterna of the RER appeared to break up into small units. The RER was tended to be less frequently coated by ribosomes. The smooth surfaced reticulum was partially absent and glycogen particles were lacking completely in most cells. In some cells, the cytoplasmic matrix was tightly packed with the increased number of mitochondria and microbodies. In group 2,the change of the hepatic cells was less prominent than in group 1.

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In order to investigate the ultrastructural changes in the hepatic parenchymal cells in albino rats fed with riboflavin-deficient diet, the electronmicroscopical study was performed. The hepatic parenchymal cells were atrophied in general area, so the cytoplasm was apparently filled with the increased number of cytoplasmic organelles such as mitochondria and microbody. In severe riboflavin deficient rats the mitochondria increased in number with occasional large mitochondria and it's out membrane appered to have a saw-toothed margin, but the intramitochondrial cristae and granules showed no changes at all. The rough surfaced endoplasmic reticulum broke up into small vesicles or tubules and ribosome disappeared from the granular reticulum. The cytolysome was never found in any hepatic cells. In hepatic cells of rat fed with riboflavin-deficient diet for 8 weeks, the degenerative change became milder and smooth surfaced reticulum increased in number near the site of confluence with the rough surfaced reticulum. It was suggested that the hepatic cell changed created by 4 weeks riboflavin-deficiency were reversible even if were kept feeding on deficient diet up to 8 weeks.

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Several morphological changes in proximal convoluted epithelial cells, characterized by presence of swelling, vacuolation of the cells and bizarre mitochondria and enlarged or sometimes vacuolated mitochondria were observed in riboflavin-deficient rats. The cytoplasmic matrix decreased extremely in its electron density. Degenerated mitochondria and cytolysome were also frequently observed in these epithelial cells. Transport vesicules and tubules beneath the microvilli were prominently decreased in number and infolding of the basal plasmamembrane was diminished as well. The crystalline inclusion bodies in the matrix of the mitochondria were occasionally found. This finding was not reported previously. In spite of these obvious changes in proximal convaluted epithelial cells, the glomerular epithelium and capillary endothelium were almost similar in apperance to those found in control rats. Other cytoplasmic organelles such as endoplasmic reticulum and Golgi apparatus, were well preserved. It was suggested that these findings were characteristic in riboflavin-deficient state.

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The ultrastructure of zona fasciculata of the adrenal cortex in rats fed with riboflavin-deficient diet was observed by electronmicroscopy. The degenerative and destructive changes were not observed in zona fasciculata of severely deficient rats. But in early stage of riboflavin-deficincy, the degeneration in the matrix and tubular cristae of the mitochondria were found. Those changes were associated with vacuolation and increased density of the matrix. The large bizarre mitochomdria infrequently appeared in the cells. Sometimes, a rod-form or queershaped inclusion was observed in the cytoplasm which has never described in the adrenal cortex and any other organ before. The presence of degenerated mitochondria and inclusion bodies were suggestive of morphological expression of the abnormal intermediate metabolic poducts that were produced by the interruption of ordinary metabolism along with riboflavin-deficiency. At 8th week of riboflavin-deficiency, the degeneration and destruction of the cells were not so severe as that was found in 6 weeks, and the lipid droplets were observed abundantly.

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It is well known that aspartate-carbamoyltransferase (ACTase) in E. coli is regulated by pyrimidine nucleotides. Since aspartate has some stimulatory effect on early thiamine synthesis in a thiazole-less mutant of E. coli and ^<14>C-aspartate is incorporated into the pyrimidine moiety of thiamine, the regulation of ACTase by thiamine or thiamine diphosphate was investigated by using a hydroxymethylpyrimidine-less mutant of E. coli. Although uracil-depletion in the mutant caused the elevation of ACTase activity about 25 fold compared with that of uracil-supplemented, hydroxymethylpyrimidine-depletion showed no effect on the activity and also thiamine or thiamine diphosphate added to the enzyme reaction mixture gave no appreciable effect on the activity. Judging from the point of physiological requirements of thiamine and pyrimidine nucleotides, it is difficult to know the relationship between ACTase and thiamine synthesizing system in E. coli.

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Mechanism of absorption, tissue distribution and metabolism of cyclocarbothiamine (CCT) in rats were investigated by using S^<35>-labeled CCT. CCT was quickly absorbed from intestine and the absorption rate reached approximately 86% within 10 minutes when 1.098mg of CCT was administered into the ligated sac of intestine. CCT was readily absorbed from the intestine, then reached the tissue where it was metabolized to thiamine. At 3 hours after injection of CCT, about 19% of total radioactivity was found in liver, 3% in kidney and 0.1 to 1% in skin, brain and heart. The radioactivity in heart remained for a longer time than in other tissues.

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Marked absorbance differences between monocyno- and dicyanoforms of corrinoids were observed at 358mμ as well as at 370mμ in the complete corrinoids and at 354mμ as well as at 368mμ in the incomplete corrinoid (cobinamide). Calculation of absorbance difference coefficients from the absorbance differences estimated at the respective two wave lengths described above gave approximately the same values for the complete corrinoids of the same molar concentrations. It is especially interesting that equal values were obtained for DBC and factor III (benzimidazolyl cobamides), and also for φ-B_<12> and factor A (adenyl cobamides). Satisfactory results were obtained from application of the absorbance difference coefficients to the differential determination of complete and incomplete corrinoids in the cells of propionic acid bacteria.

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It was observed that formation of desthiothiamine through the desulfurization of thiamine was remarkably increased in the presence of glycine or other twelve amino acid, i.e., α-alanine, valine, β-alanine, γ-aminobutyric acid, ε-aminocaproic acid, aspartic acid, glutamic acid, lysine, ornithine, arginine, serine and taurine with equimolar NaOH. Desthiothiamine was obtained in 78% yield when thiamine-HCl (1.55×10^<-2> mole) with equimolar glycine in a trimolar NaOH aqueous solution was incubated at 20℃ for three days. In the presence of other amino acid, desthiothiamine was obtained in 20 to 67% yield after the incubation for six days at 20℃. It was shown to be optimal to yield desthiothiamine that NaOH in 3.0 molar equivalents to thiamine-HCl with glycine or α-alanine, in 2.67 molar equivalents with β-alanine, and in 4.0 to 4.33 molar equivalents with aspartic acid were incubated for three or six days. Decrease in yield of desthiothiamine was observed when the above desulfurization of thiamine with amino acid was carried out at higher temperature above 60℃ for three or six hours.

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It was observed that yield of hydroxyiminothoiamine (IV) was increased by addition of glycine with equimolar NaOH in the reaction of thiamine (I) with hydroxylamine in NaOH solution. The addition of desthiothiamine (III) also increased the yield of (IV), corresponding to the amount of (III). When desthiothiamine was reacted with hydroxylamine, phenylhydrazine, ammonia or benzylamine in weak acidic or alkaline solution, formation of hydroxyiminothiamine, phenylhydrazonothiamine (V), imidazolethiamine (VI) and benzylaminothiamine (VII) were demonstrated by means of paper partition chromatography. When desthiothiamine, hydroxyiminothiamine, phenylhydrazonothiamine or thiosemicarbazonothiamine (VIII) was heated with 10 % hydrochloric acid, formic acid was demonstrated in the reaction mixture, and thiamine was also shown to be formed by saturation of H_2S in the acetate buffer solutions (pH 5.0) of these compounds at 90℃. But the formation of formic acid and thiamine were not demonstrated in the case of imidazolethiamine and benzylaminothiamine.

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Since dihydrothiamine (DHT) has been assumed as an intermediate or a precursor in the process of thiamine biosynthesis, the existence of DHT-like substances in leaves of some ascorbic acid-rich plants, mainly of Iridaceae, was examined. Samples were homogenized with 60 % ethanol or 2 % solution of metaphosphoric acid under ice-cold condition using a Waring blender, followed by centrifugation to separate the supernatant, in which the thiamine was measured by usual cyanogen bromide method before and after the oxidation treatment with 2,6-dichlorophenolindophenol. The amount of the DHT-like substances was calculated by substracting thiamine contents before the oxidation from that after the oxidation. In only two (Iris tectorum Maxim and I. pseudoacrorus L.) out of eight kinds of plants tested, less than 5〜6μg % of DHT-like substances were found, but in others not at all. DHT was more stable in homogenate than in equeous solution, but ascorbic acid had no ability to improve the stability of DHT in its aqueous solution at pH 4.0 or 7.3,showing inversely a tendency to accelerate the decomposition of DHT.

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The oxidation of dihydrothiamine (DHT) with various metal ions, ascorbic acid and hydrogen peroxide was studied as a model experiment for thiamine formation from DHT under a physiological condition and the following results were observed. Only cupric ion among thirteen kinds of metal ions tested showed some activities for the oxidation of DHT in a solution at pH 7.3. The activity of cupric ion was seemed to be due to a catalytic action toward the oxidation of DHT by molecular oxygen, because more thiamine was produced from DHT at the presence of oxygen than nitrogen or carbon dioxide and thiamine formation decreased proportionally with the increase of cupric ion. The coexistence of ascorbic acid or hydrogen peroxide with cupric ion which appeared to rise such free radical as OH・did not cause any remarkable thiamine formation. On the other hands, the coexistence of cupric ion and dehydroascorbic acid showed apparently more formation of thiamine than cupric ion or dehydroascorbic acid alone. This suggests that if DHT exists in biological bodies, it would be easily converted to thiamine with combined action of dehydroascorbic acid and metal ions.

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Papain, when preincubated with α-benzoylarginylamide (BAA) or α-benzoylarginine ethyl ester (BAEE) at pH 4.0,0℃ for 1 hour, was unable to react with thiamine propyl-disulfide (TPD) to liberate thiamine. If preincubation was undertaken at pH 8.5 or 9.0,papain was able to liberate thiamine from TPD. It was suggested that the acyl-thiol enzyme was formed in the preincubation to prevent the reaction with TPD. The acyl-thiol enzyme in preincubation with BAEE was most stable at pH 4.0,0℃ or even at 40℃ but the acyl-thiol enzyme with BAA was less stable. After preincubation of papain with BAEE and precipitation by trichloroacetic acid, benzoylarginyl papain was shown to contain 0.6 mole benzoylarginine per mole of papain by hydrolysis at pH 4.0,60℃.

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The nicotinamide coenzyme contents in the livers estimated fluorometrically after administratio of L-tryptophan intra-peritoneally were found to be about the same as normal in alloxan-diabetic rats, although the urinary excretion of N'-methylnicotinamide in alloxan-diabetic rats was about one-half of that in normal rats. In order to know the metabolic rate of NAD synthesized in the alloxan-diabetic rat liver, the increase of NAD content in the liver and the urinary excretion of N'-methylnicotinamide after administration of niacin were compared between both rats. The each values estimated had no difference between normal and alloxan-diabetic rats. The liver NADase activity was also same in both rats. The mechanism maintaining NAD in the diabetic rat liver at normal level was discussed.

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