The oral administration of dl-α-tocopherylnicotinate (900mg, daily) to 21 hypercholesterolemic patients for 7-40 days was resulted in 14% decrease (p<0.001) in the serum total cholesterol levels with improvement of the subjective symptoms. Feeding of dl-α-tocopherylnicotinate or dl-α-tocopheryl acetate (as 0.24% dl-α-tocopherol) to mice for 21 days caused a decreased incorporation of acetate-1^<14>C into hepatic cholesterol. When dl-α-tocopherylacetate was injected to mice at a dose of 200mg/kg every five days for 60 days, the drug caused a decreased incorporation of acetate-1^<14>C into hepatic cholesterol, but had no effect when mevalonate-2^<14>C was used. When mice were fed a basal diet with 0.24% dl-α-tocopherylacetate for 21 days and cholesterol-4^<14>C was injected, the drug caused an increase of the fecal excretion of ^<14>C, decreases of hepatic cholesterol-^<14>C and the ratio of liver to blood free cholesterol specific activity derived from cholesterol-^<14>C. The results suggest that α-tocopherylnicotinate may produce serum cholesterol-lowering effect by inhibiting the biosynthesis of cholesterol and increasing the fecal excretion of cholesterol.

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Since many defective behaviors in breeding were found in mice fed a diet, an opportunity arose to check the diet. From the consideration for the composition of this diet, it was supposed that this diet might be deficient in vitamin E. Investigation was made on the role of vitamin E in relation of the reproductive performance of mice, and it was confirmed that this diet was lacking for vitamin E. In this experiment, it was clarified that vitamin E deficient female mice cannot breed normally, and the fetal death with resorption occured after the 12th day in gestation period in many cases. Moreover, anomalies, e.g.exencephalocele, anophthalmia, microphthalmia, irregular cheilosis and edema, were observed in many of the survived fetuses. These sterilities were completely prevented by the addition of α-tocopherol.

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The application of 2,6-dichlorophenolindophenol, an oxido-reducing indicator, to a method for the microbiological cup-plate assay of thiamine with Lactobacillus fermenti was effective for enhancing the contrast between the growth zones of the organism and the area of non-visible growth. The results obtained by this technique were further compared with those of the original method. The zones of exhibition were reproducible and there was no significant difference in their sizes between both methods. The highest probable error was a little higher than the original assay method. This indicator may be available for increasing the visibility of the growth zones without a great lowering of the precision.

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A difficulty to obtain a clear and sharply defined growth zone of the test organism has been reported in the cup-plate method for bioassay of certain vitamins. Thus, the application of resazurin, an oxido-reducing dye, used as an indicator, to the cup-plate procedure was attempted to increase the visibility of the growth zones of Lactobacillus fermenti on the assay plate for thiamine. Under the aerobic condition the dye was used after the conventional plate procedure and it was reduced to colourless substance within several minutes by the organism. There was a better contrast between the growth zones and the dark violet zone of back ground. The growth zones observed in this manner coincided accurately with the original zones and it was found from the statistical analysis of the results for thiamine that resazurin treatment scarcely affected the precision of the plate method.

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Cup-plate technique as a microbiological assay method for vitamins has not yet been brought to practical use. A fundamental study of a plate-assay procedure for folic acid with Streptococcus faecalis as the test organism was undertaken in view of several advantages in the vitamin estimation. The folic acid content of powdered agar added to basal medium affected the clearness and sharpness of the growth zones. There was the linear relation between the diameters of growth zones and the logarithms of the concentrations within the range of 10 to 4000 mμg/ml of pure folic acid solution, and in the statistical evaluation of the assay results the maximum errors of the estimate were below 15%. These results suggest the possibility that the cup-plate method may be available for the microbiological assay of folic acid.

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The teratogenic effects of pantethine were investigated in ddYF mice and Wistar-Imamichi rats. Pantethine was given orally at dose levels of 600 and 60 mg/kg in mice and of 600,300 and 60 mg/kg in rats for consecutive 7 days from 6 to 12 days of gestation. No teratogenic action of pantethine was found on fetuses and newborns both in mice and rats. The accelerative effect of pantethine on development of fetuses was observed both in pregnant mice and rats.

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It was reported by Esselen and Fuller that a certain strain of E.coli has the activity of reducing dehydroascorbic acid. Authors first observed dehydroascorbic acid reductase in E.coli K_<12>. The enzyme was effective on hydrogen donors such as glutathione, homocysteine, thioglycolate and cysteine, but not effective on reduced NAD, reduced NADP, CoA and lipoic acid. Recently, authors observed that as long as pyruvate exists in the medium containing E.coli, dehydroascorbic acid is reduced. As this reducing activity is independent of dehydroascorbic acid reductase, a new pathway has been suspected. As this reducing activity is dependent upon a pyruvate oxidase system, dehydroascorbic acid is assumed to be reduced into ascorbic acid following reaction sequences in the bacterial cells. The same reactions were found in pig heart muscle.

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TBA-value of the liver of rat administered with hardened oil is higher than that with soy bean oil. When 2.0mg of α-tocopherol was administered per day to a rat, TBA-value of the liver was lowered in the above-mentioned two cases.

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Rat was fed with soy bean oil or cod oil containing diet for 2.5 months α-Tocopherol accelerated the body weight gain, except in the case of highly oxidized cod oil. Administration of α-tocopherol decreased the TBA- value as well as the peroxide value of fat extracted from the rat. The TBA-value was in parallel to the peroxide value.

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HeLa cells were cultivated in thiamine-free TC Medium Eagle-HeLa containing 10% bovine serum and thiamine hydrochloride or various thiamine derivatives. Effects of these compounds on growth of the cells were examined by use of counting the nuclei. The thiamine derivatives tested were as follows : Thiamine phosphates (Metabolase for injection, TMP and TDP) ; symmetric disulfides of thiamine (TDS and BTDS) ; asymmetric disulfides of thiamine (TPD, TED, TMD, TTFD, TOED and TAD) ; S-acyl derivatives of thiamine (DAT, DBT and BTMP) ; S-alcoxycarbonyl derivatives of thiamine (CET and DCET). All of them stimulated growth of the cells at low concentrations. Especially, Metabolase, BTDS, TPD, TAD, TOED, DAT, DBT and DCET proved to have greatly stimulating effects on it at low concentrations from 0.005 to 0.5μg/ml. Asymmetric disulfides, especially TPD, TMD and TED had strikingly inhibitory effects at the highest concentration employed (50μg/ml), those of TTFD and TOED were not so remarkable, and those of TAD were the weakest. On the other hand, the inhibitory effects of the others were scarcely detected even at the same concentration.

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Cells obtained from various sources were cultivated in growth media containing high concentrations of disulfide derivatives of thiamine, such as TPD, TTFD or BTDS. Effect of these derivatives on growth of the cells were investigated by counting of nuclei. The cells employed were as follows : HeLa cells, L cells, hamster kidney cells in primary and secondary monolayer cultures, and monkey (Macacus cynomolgus) kidney cells in primary monolayer cultures. Although growth-inhibitory effects were scarcely detected at concentration of 5μg/ml of TPD, cytopathic and growth-inhibitory effects were remarkable at concentrations higher than 30μg/ml of TPD. However TTFD did not show such notable effects as TPD did, furthermore, those of BTDS were the weakest at the same concentration. The results obtained above indicated that the similar effects of these derivatives were always found in any monolayer culture not only on normal cells, but also on malignant cells, on both sorts of cells whether they were epithelial or nonepithelial, moreover on mammalian cells generally. But growth of chick embryo heart tissues in plasma clot cultures was not checked even by high concentration (100μg/ml) of TPD.

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In order to examine effects of decomposition products which might be produced by cysteine from TPD, HeLa cells were cultivated in TC Medium Eagle-HeLa containing high concentrations of TPD and cysteine. Only remarkably cytopathic and growth-inhibitory effects as strong as those of TPD alone were found. In the following experiment effects of propylmercaptocysteine on growth of the cells were investigated by means of cultivating the cells in the media containing the compound by use of counting the nuclei, and such striking effects as those of TPD were not proved by the compound even at high concentrations (30-50μg/ml). So it seemed that the checking effects of TPD were not based on propylmercaptocysteine alone from the results. Finally monolayer sheets of HeLa cells, L cells and monkey kidney cells were maintained in maintenance media containing each derivative so that effects of disulfide derivatives of thiamine, such as TPD or BTDS, at high concentrations (30-50μg/ml) on maintenance of cell sheets and on survival of the cells were observed. Any sheet was scarcely damaged by BTDS, furthermore the checking effects of TPD were not striking on it, although TPD had the notably inhibitory effects on the cells in floating condition in early stage of monolayer cultures at the same concentrations.

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Depending upon reaction temperatures, the reaction of thiamine with phosgene in alkaline solution gave rise to two different compounds as follows. At 0-5℃, N-[1-(2-oxo-1,3-oxathian-4-ylidene) ethyl]-N-[(4-amino-2-methyl-5-pyrimidinyl) methyl]formamide (V) and at -20℃, bis-2-[N-(4-amino-2-methyl-5-pyrimidinylmethyl) formamido)-1-(2-hydroxyethyl) propenethiol carbonate (XVI). The structure of XVI could be established by the fact that the same compound was formed in the reaction of phosgene with O-tetrahydropyranylthiamine-Na followed by hydrolysis with hydrochloric acid. The structure of V was confirmed by UV, IR and NMR spectroscopies and by the observations that the treatment of V with NaOR, NaSR and amines produced O-carbalkoxythiamine, O-carbthioalkylthiamine and O-carbamoylthiamine, respectively. Similarly, hydroxyethylthiamine gave N-[1-(2-oxo-1,3-oxathian-4-ylidene) ethyl]-N-[(4-amino-2-methyl-5-pyrimidinyl) methyl]lactamide (VII) in the same way. Thermal decomposition of V was investigated. These new compounds showed thiamine-activity, and V is particularly remarkable among them, since it was quite readily absorbable from the intestine.

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Studies were carried out on the physicochemical properties of cyclocarbothiamine (CCT), a new thiamine derivative developed in this laboratory. The compound was obtained as white crystal or cystalline powder with bitter taste. The ultraviolet spectrum of the compound has its absorption maximum at 243 mμ in acidic solution, which varies depending on pHs of the solution. The infrared spectrum shows specific absorption bands at 1685-1690cm^<-1> (νc=o) and 1175cm^<-1>(νc-o-c) The compound was found to be stable in the form of crystalline powder or powder mixtures ; it is also more stable in acidic solution than in neutral or alkaline solutions

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Cyclocarbothiamine (CCT), a new thiamine derivative was proved to possess biological potency for the growth of rats. Administration of thiamine-HCl or CCT resulted the similar effects in the level of thiamine in rats organs and on recovery of transketolase activity. Thiamine-HCl added in blood in vitro showed no effect on the transketolase activity. When CCT was added to a suspension of blood cells in 0.9% NaCl solution and incubated for 30 minutes at 38℃, recovery of the enzyme activity was observed. CCT may be converted to thiamine pyrophosphate under this condition. When massive doses are given per os, CCT posses advantage over thiamine-HCl in the absorption rate in man.

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Clinical effects of homopantothenic acid (HOPA) were studied in 20 patients with various neurological diseases, consisting of epilepsy, parkinsonisms, tremor of various origins, tic, myoclonus and organic brain syndrome. Beneficial effect was obtained in 4 out of 5 idiopathic epilepsy patients, 2 of tremor and 1 of tic. Suppression of paroxysmal discharge and normalization of basic rhythmus were also noted electroencephalographically after administration of HOPA.

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The anticonvulsive effects of homopatothenic acid (HOPA) were examined on cases with refractory epilepsy, consisting of 5 cases with infantile spasms, 2 cases with absence, one case with akinetic jerk and 6 cases with grand mal epilepsy. HOPA was given orally amount of 1.0-1.5 g per every day for 2-8 weeks. Following the therapy, dramatic responses were observed on 6 out of 9 cases with petit mal type, but discouraging effects on 5 out of 6 cases with grand mal type. The effects seemed to be transient because of reappearance of seizures after the further therapy. It was noteworthy that HOPA had an anticonvulsive effect on some of epileptics resistant to the other drugs.

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