Colorimetric determination of homopantothenic acid, D(+)-N-(α, γ-dihydroxy-β, β-dimethylbutyryl)-γ-amino-butyric acid was described. Homopantothenic acid is hydrolyzed to γ-aminobutyric acid (GABA) in alkaline solution and its amino-nitrogen is determined colorimetrically at 465mμ with sodium 1,2-naphthoquinone-4-sulfonate at pH 8.8〜9.6. If GABA, one of decomposition products is contaminated, it can be eliminated by Amberlite CG-120 and then the above procedures can be applied.

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Physicochemical properties of Ca-homopantothenate was investigated. It is easily soluble in water, but insoluble in organic solvents, only slightly soluble in methanol. Apparent dissociation constants obtained by titration curve was 4.52. Rf is 0.86,when a developing solvent con-sisted of n-buthanol・acetic acid・water is used, by the thin layer chromatography. Infrared absorption spectrum was supported its structure. Ultraviolet absorption spectrum of its aqueous solution has no peak above 2O0mμ. Stability of its aqueous solution on pH profile was estimated by the kinetic technique. The most stable pH ranges were 5.0〜6.0.

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The effects of homopantothenic acid (HOPA) on the brain were invetigated electrophysio-logically. Local application and intra-arterial administration of the drug did not affect the trans-callosal potentials of the cats anesthetized with sodium pentobarbital. This suggests that the action of HOPA in the central nervous system does not depend upon γ-aminobutyric acid released from HOPA itself. The spontaneous EEG of the unanesthetized rabbits restrained stereotaxically was slightly changed into the alert pattern by HOPA. The intra-venous administ-ration of 500 mg/kg of HOPA did not antagonized pentylenetetrazol or picrotoxin induced seizures, but when 5 mg/kg of HOPA was administered intra-cisternally, these seizure discharges were arrested. The fact shows that HOPA hardly passes through the blood brain barrier.

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On comparing the uptake of homopantothenic acid (HOPA) and pantothenic acid (PaA) into brain of rat after intraperitoneal injection, it was shown that HOPA penetrated into the central nervous system as readily as PaA did. The penetration of HOPA into eight regions of rat brain has been measured. The marked regional differences were not observed and pre-treatment with hydroxymethylpyrimidine produced little change in the concentration of HOPA in brain regions. The experimental data of the accumulation of HOPA and γ-aminobutyric acid (GABA) by brain slices suggest that HOPA is passively transported by brain in vitro while GABA is actively transported. Almost all of HOPA orally administered was excreted in urine by mice in 24 hours. More than 95 percent of urinary metabolites was HOPA and less than 5 percent was an unknown metabolite.

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Safety evaluation of homopantothenic acid (HOPA) was made by chronic toxicity and teratolo-gical tests. In the chronic toxicity test, HOPA was administered to rats orally in doses of 2 or 1g/kg daily for six months. The general condition of these animals was optimal throughout the experimental interval ; weight gain, hematologic determinations, gross and microscopic findings did not suggest the occurrence of drug-related abnormalities. In the teratolo-gical test, 2 or 1 g/kg of HOPA was given orally to pregnant rats and mice every day from the 7 th to the 14 th day of gestation. No influence of HOPA was observed on both fetuses and newborns obtained from the mother animals.

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A histopathological observation has been performed on the lesions of rats which were administrated with the dosis of 400 mg/kg or 100 mg/kg of homopantothenic acid during 3 months. No pathologic change caused by the acid was encountered in the heart, lungs, spleen, liver, pancreas, stomach, intestines, kidneys, hypophysis, thyroid gland, adrenals, brain and testes.

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69 off-springs were obtained from 10 rats which were administrated with 400 mg/kg of homopantothenic acid from the 2nd day to 21st day of pregnancy. No malformated newborne was obtained in this series. Only 2 of 69 newbones were died during lactation.

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Homopantothenic acid was given orally in 28 mentally retarded children ranging in age from 5 to 11 years. An improvement was noted in study desire, behavior and speech in some and there was a rise in intelligence quotient of more than 6 positive in about one-third of the cases. Disappearance of slow waves, increase in a waves and appearance of basic pattern was observed electroencephalographically. Side effects were not observed during or after administra-tion.

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Pharmacological effects of O-butyrylthiamine disulfide (BuTDS) were studied in mice, rats, rabbits and dogs. BuTDS markedly prolonged the forced swimming time of mice. BuTDS reduced the development of the rat pad edema induced by serotonin, but exerted no significant effect on the formalin edema. BuTDS caused a blood pressure fall and a slight depression of the cardiac movement in large doses. These effects of BuTDS were less marked as compared to those of thiamine-HCl. BuTDS increased the tonus and movements of the rabbit intestine in situ, while it, on the contrary, reduced the motility of the isolated rabbit intestine. Contractions of the pyloric sphincter of the dog were increased in magnitude as well as in frequency, whereas the tone of the cardial region was slightly reduced by BuTDS.

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A favorable condition was reexamined for the determination of thiamine by cup-plate method using Lactobacillus fermenti. The size and sharpness of periphery of zone were influenced by various biological and physicochemical factors, such as pHs of medium and thiamine solution or times of diffusion of test solution into the plate. Therefore, the procedure must be done under a definite condition. The following condition was preferred by the authors. Assay medium : Sarett・Cheldelin's with 1% agar, 1.5mm plate thickness, pH 5.7〜6.1. Thiamine solution : pH 5.6〜6.0. Incubation : 37℃, 16〜18hrs.

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The cup-plate method was proved to give a sharp and clear growth zone of Lactobacillus fermenti for thiamine determination. It was recognized a linear relationship between the diameter of growth zone and logarithm of the concentration of thiamine over a wide range of 0.1 to 1Oμg per ml. The statistical analysis of the results revealed that the maximum error of cup-plate method using 5 plate mounted only to 20%, and it is supposed to give most reproducible result among other methods, though it is somewhat inferior in sensibility. However, in case of its application to biological materials, a precaution should be taken against the possible existence of interfering factors in them.

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Some pharmacological effects of pantethine (PTSS) were examined. When PTSS was injected to rabbits intravenously, no change was found both in b1ood pressure and respiration in doses of 5 to 20mg/kg. PTSS was ineffective on the isolated intestine of rabbit and guinea pig, the isolated uterus of rat and guinea pig, and on the isolated vas deferens of guinea pig. Effect of acetyl choline, histamine, nicotine, and serotonin on the intestine and that of epirenamine on the vas deferens were not antagonized by PTSS. PTSS was ineffective also on the musculus rectus abdominis of frog.

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Thiamine-ylide (II) obtained by the treatment of thiamine-HCl (I) with piperidine reacted with diethylbenzoylphosphonate (III) to give 1-phenyl-3-(2-hydroxy) ethyl-4,9-dimethyl-1,6-dihydropyrimido[4', 5'-4,5]pyrimido[2,3-c][1,4] thiazine (IV), IV-benzoate (V), and N-benzoylpiperidine (VI), while the reaction of morpholine treated I with III gave only small amount of IV, but N-benzoylmorpholine (VII), and 4-(2-methyl-4-amino-5-pyrimidyl) methylmorpholine (VIII) as main reaction products. It is interesting that the evidence thus obtained VIII from the above mentioned reaction offers a new progressive demonstration to previous amine substitution reactions to thiamine-thiazole nucleous in the presence of sulfite anion.

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The thiamine content in soybean seedlings increased in vivo by administrating both moieties the pyrimidine and thiazole, and the amount of the vitamin synthesized increased with the concentrations of two moieties supplied. When 2-methyl-4-amino-5-hydroxymethylpyrimidine and thiazole-^<35>S were used, the amount of the vitamin formed which was assayed by the usual thiochrome method was good agreement with that which was calculated from radioactivities of the thiamine-^<35>S synthesized. The vitamin was mainly synthesized in the cotyledon of seedlings in either the light and dark.

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It was found that thiamine was biosynthesized by green leaves from 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate and thiazole phosphate or thiazole in the presence of MgCl_2. Some properties of the enzyme reaction were investigated with the homogenate of radish leaves. The enzyme had an optimum pH at around 9.0 and had the maximum activity at 30℃. The production of thiamine in the homogenate was inhibited abruptly after an elapse of the reaction for about 30 minutes, and then the vitamin formed was destroyed gradually. The destruction of the vitamin during the incubation was protected partly by the addition of mercaptoethanol.

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The previously reported growth factor (tentatively named as Factor K) for Streptococcus faecalis 10Cl was extracted from the dried mycelium of Aspergillus oryzae (1kg) with a sulfuric acid solution and concentrated about 2400-fold with a yield of 9.2% by means of chloroform・water extraction and Amberlite IR-4B resin treatment.

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From our laboratory, a differential assay method of vitamin B_6 group was already reported, involving the separation of PIN (pyridoxine), PAL (pyridoxal) and PAM (pyridoxamine) using a cation exchange resin (Duolite-C 60), and microbiological assay procedure with Saccharomyces carlsbergensis. But this resin has not been commercially available, so the reexamination of new resin and a differential assay method of vitamin B_6 group containing the phosphoric esters in addition to free forms have been reported in this paper. The general method is as follows. First, the extracts of natural products are added on the anion exchange resin, Dowexlx8 (AcO^-), the phosphoric esters of vitamin B_6 are adsorbed on the resin, while free types are not. The latter is then added to the column of the cation exchange resin, Amberlite IRC CG-50 (H^+, 100〜200 mesh). PIN and PAL are eluted with acetate buffer, pH 4.6,while PAM is retained. The fraction containing PIN and PAL are divided in two parts, and PAL in one part is inactivated by acetone-alkali treatment. PAM adsorbed on column is eluted with acetic acid. Each content of PIN, PAM and sum of PIN and PAL are determined by the microbiological assay with Sacch. carlsbergensis. On the other hand, the phosphoric esters adsorbed on the Dowex-1 are eluted with acetic acid, hydrolyzed to the free forms and treated as described above. The expriments with authentic vitamin B_6 samples and some natural products (chick liver, baker's yeast etc.) under this method gave the satisfactory result. The effect of salts and limiting concentration of vitamin B_6 compounds in this assay are also showed.

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This paper deals with so called red-yellow shift, the color change of corrinoid compounds caused by pH change. The relation between absorption spectrum and electronic charge in cyanocobalamin, monocyano-monoaquocobinamide, methylcobalamin and monomethyl-monoaquocobinamide was examined in a range of pH 1〜11. A spectral change accompanied with a charge change was found to occur at pH 2.5〜3.5 and at pH 8〜9 respectively in methylcobalamin and in monocyano-monoaquocobinamide, while cyanocobalamin was always neutral and red in the whole pH range, and monomethyl-monoaquocobinamide was always positive and yellow above pH 3. Although the results described above suggested that the yellow color was due to the protonation, monomethyl-monothiocyanatocobinamide was found to be neutral in spite of its yellow color, the absorption maximum being at 475mμ. Considering that the absorption maximum of monomethyl-monopyridinocobinamide and monomethyl-monocyanocobinamide was at 523mμ and at 580mμ respectively, it is concluded that the visible absorption spectrum of a corrinoid compound with a certain upper ligand depends upon the lower ligand very much. The color change caused by protonation can be thus explained as a result of a replacement of the lower ligand.

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It is widely accepted that cyano- and/or hydroxo-cobalamin can bring forth a complete remission of haematologic alterations in pernicious anemia, but the neurologic disorders can not necessarily be cured completely with either vitamin B_<12>. Since vitamin B_<12> is expected to be converted to coenzyme form, 5,6-dimethylbenzimidazolyl cobamide coenzyme, which is now believed to be a naturally occurring active vitamine B_<12>, it may be reasonable to assume that conenzyme B_<12> might be more effective for treatment of such neurologic disorders. With this in mind, therapeutic trial with coenzyme B_<12> was made in neurologic impairments associated with various diseases, in order to observe the effect on neurological symptoms. As a result, more than was expected, a marked therapeutic effect of coenzyme B_<12> was observed in 10 of 12 cases tested, most striking among which was a case of myelitis, in that cyano- and/or hydroxo-cobalamin was found ineffective despite massive doses of total 136 mg were parenterally given. This paper describes the results of this observation.

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Erythrocyte count and Sahli's value increased about 27 and 23%, respectively, after interavenous administration of thiamine tetrahydrofurfuryldisulfide 100 mg daily for 20 days on 10 normochromic anemic patients. However, leucocyte-, platelet- and reticulocyte count and also serum iron content were not influenced.

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