The present study was undertaken to see the curative effects of cyclocarbothiamine on neurological disturbances of diabetic patients. Twenty five diabetic patients were selected for the study. These patients had subjective and/or objective neurological signs, such as neuralgia, paresthesia, hypesthesia, absence of knee jerk, decreased motor nerve conduction velocity, etc. Most of the cases received a daily doses of 150 mg of cyclocarbothiamine for 6-60 days. In a few cases the daily doses was increased to 225mg. The curative effects were judged by the disappearance of subjective symptoms and also by the changes in motor nerve conduction velocity. By the treatment, an improvement or a disappearance of subjective distress was obtained in 18 of the cases and an increase of motor nerve conduction velocity was seen in 12 of the 25 cases.

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The effects of phytic acid and its salts on the growth of various strains of Lactobacillus acidophilus was investigated. It was clarified that phytic acid, Ca-phytate and CaNa-phytate accelerate the growth and acid production of the bacillus in small quantity while inositol did not.

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Method of separation and determination of tocopherols in soybean oil was investigated by gas chromatography, equipped with a hydrogen flame ionization detector. The petroleum ether solution of unsaponifiable matter of soybean oil was transfered to Florisil column and tocopherols were eluted with ether. After removal of sterols by methanol and 80% ethanolic solution of digitonin, they were acetylated and injected into gas chromatograph. Gas chromatographic analysis was carried out on the column packing consisted of Chromosorb W coated with 1.5% SE-30 at a column temperature at 250℃. By the method, tocopherols in soybean oil were satisfactorily separated and determined.

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Studies on the determination of fatty acid esters of ascorbic acid (AsA) contained in oily preparations were made. Alkaline solution is usually used for hydrolysis of the esters of fatty acid, however, it was not suitable for the determination of fatty acid esters of AsA, because oxidation of AsA was enhanced during the hydrolysis. But the decomposition of AsA was also observed during the hydrolysis with concentrated acids. Ascorbic acid monostearate (AsA-MS) or ascorbic acid dipalmitate (AsA-DP) was found to be completely hydrolysed in 2% hydrochloric acid in ethanol. The hydrolysate of esters showed blue color by 2-nitro-4-methoxyaniline method as well as AsA. AsA-MS itself also showed the color without hydrolysis. Using this method, 95% recovery was obtained for AsA-DP in a vanishing cream.

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The oxidation of 3-hydroxybutyrate by liver mitochondria of normal guinea pigs and of choline deficient ones were compared. The enzymic activities of intact and sonic disrupted mitochondria of choline deficient animals were found to be as less as 74 percent of those of normal animals. The cause of these low activities was not considered to depend upon NAD level of mitochondria. The differences of P : acetoacetate ratio and P : O ratio were not significant.

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Palmitate oxidation by liver mitochondria was studied with normal and choline-deficient guinea pigs. The oxidation of palmitate by liver mitochondria was largely activated by the addition of carnitine in vitro, especially, the formation of acetoacetate was highly increased. In the case of choline-deficient animals, the formation of acetoacetate from palmitate with addition of ATP was significantly increased, however, oxygen uptake was low. The calculated value of palmitate consumption of choline-deficient groups was close to that of normal ones. The effects of some coenzymes, i.e. ATP, cytochrome c and CoA, on palmitate oxidation were also discussed.

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The reaction of β-lipoic acid with acetic anhydride under mild conditions yielded an unknown product which liberated acetic acid by hydrolysis with NaOH. The product showed almost the same microbiological activity for Streptococcus faecalis 10C1 and Corynebacterium bovis as α-lipoic acid and β-lipoic acid. The results of ultraviolet and infrared spectrophotometry and other tests confirmed the identity of the acetylated product with an acetoxy derivative of α-lipoic acid, the formation mechanism of which is thought to be analogous to that of Pummerer reaction. Deuterium exchange studies revealed the possibility of a proton attracted to the carbon adjacent to the sulfoxide group in β-lipoic acid. A possible steric hindrance caused by the bulky alkyl side chain would support that 8-C-acetoxylipoic acid is derived from β-lipoic acid having 8-sulfoxide structure. Associated with the competitive inhibitory effect of 8-C-methyllipoic acid, the results obtained in this experiment may suggest the possible reactivity at the C-8 position of β-lipoic acid which may play an unknown role in biological systems.

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When α-lipoic acid in chloroform was oxidized with 5 molar ratio of t-butyl hydroperoxide for 2 days at 38℃, an unidentified lipoic acid derivative (X) was detected at Rf 0.90 on the paper chromatogram using 2.8% aq. ammonia saturated n-butanol as a developing agent. The zone corresponding to X was extracted with methanol and the extract was purified by paperor thin layer chromatography. X was obtained as a yellow oily substance in about 5% yield. It was positive to KCN・nitroprusside reagent and exerted an inhibitory effect on Streptococcus faecalis 10 C1. Its spectrum indicates that X is t-butyl ester of lipoic acid sulfone. Free acid (Y) was obtained by hydrolysis of X in ca. 4% yield. The infrared spectrum of Y was essentially identical with that of α-lipoic acid except that the peaks assignable to a sulfone group were observed at 1310 cm^<-1> (ν_<as>SO_2) and 1140 cm^<-1> (ν_sSO_2) . Its elementary analysis value corresponded to that of monosulfone. Y showed an inhibitory effect on Stc. faecalis 10 C1 with a 50% inhibition index of about 100.

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Enzymatic method based on sorbyl-CoA formation (Wakil and Hubscher, 1960) was applied to the rat liver extracts for determination of CoA. The stability of CoA and the procedure for extraction of CoA from the rat liver were examined. The following procedure for preparing the liver extract was chosen. Liver homogenate was boiled immediately for 7 minutes, then centrifuged in the cold temperature. The supernatant was adjusted to pH 4.0 and centrifuged after boiling. The supernatant was used for determination of CoA after adjusting pH to 9.0. CoA values obtained by sorbyl-CoA formation were shown to be about a half of those obtained by the sulfanilamide method or of conjugated pantothenic acid values by microbioassay method. These results suggested that there were considerable amounts of bound form of pantothenic acid in the rat liver, besides CoA.

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Biosynthesis of CoA from 4'-phosphopantetheine was studied, in vitro, and it was confirmed that the latter is almost completely converted to CoA by an enzyme, partially purified from rat liver homogenate, according to the procedure of Hoagland and Novelli, while formation of CoA from pantetheine, pantothenic acid or 4'-phosphopantothenylcysteine was not observed. When liver extract was incubated with addition of the enzyme, the coenzyme value increased about 2 times of that of before incubation, which indicates that precursors of CoA is present in rat liver in almost same level as CoA. The amount of CoA and its precursor in rat liver is observed to run parallel with growth from birth up to 30 days.

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Experiments were done on the biosynthesis of CoA from pantothenic acid, in vivo. Young albino rats were fed on various levels of pantothenate or pantethine as 0,0.45,2.3,9.2,23.0 and 230.0 μ moles supplementation per 100g of pantothenic acid deficient diet. Hepatic CoA and its precursors (dephospho-CoA and 4'-phosphopantetheine), bound and free pantothenic acids were determined. 1) When rats were fed on deficient diet, both CoA and its precursors in liver decreased. 2) The liver CoA content was highest when deficient diet was supplemented with 9.2 μmoles per 100g of pantothenate or pantethine. 3) When the deficient diet supplemented with 2.3-23.0 μmoles of pantothenate or pantethine was administered, the contents of precursors in liver were almost equal to those of CoA. Such a finding that rat liver contains considerably larger amount of CoA precursors is quitely different from Novelli's conclusion in which he mentioned that almost all the bound pantothenic acid was CoA itself. 4) No difference between the liver CoA contents of pantothenate and of pantethine-supplemented groups, but the ratio of CoA to total pantothenic acid was always higher in pantethine-supplemented groups.

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The influence on skeletal muscle fatigue by the massive administration of thiamine-HCl and thiamine tetrahydrofurfuryldisulfide (TTFD) was studied in the rabbits experimentally, and the following results have been obtained. About neuro-muscular transmission and arterial pressure, the effect of TTFD was different from that of thiamine-HCl. In a certain period of time after injection of TTFD 5 mg/kg interavenously, the amplitude of ankle motion on kymograph caused by indirect stimulation was increased, but that was not obtained by the injection of 5 to 7 mg/kg of thiamine-HCl and 5 mg/kg of ATP. The effect perceived through direct stimulation was not evident as through indirect stimulation, even though the equal dosage of TTFD was administered. Therefore, it is presumed that TTFD acts on nerve rather than on muscle.

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Coagulation time (beginning, end) and bleeding time were reduced about 22,4 and 25%, respectively, after intravenous administration of thiamine tetrahydrofurfuryldisulfide 100 mg daily for 20 days on 10 normochromic anemic patients. However, homolytic reaction, prothrombin time, liver function and also renal function were not significantly influenced.

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The calcium salt of homopantothenic acid was given orally in 10 cases of postencephalitic sequelae and the therapeutic effect was examined clinically and electroencephalographically. The dosage was 1〜3 g daily for 14〜169 days. The treatment was very effective in 3 cases, effective in 6 and ineffective in 1 case. Electroencephalographically, restoration of the basic waves to a pattern normal for the age was noted parallel with the improvement in the clinical symptoms. Side effects were not observed.

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