The direct injection of semicarbazide (SC), an antivitamin B6 (anti-B6), into the lateral ventricle of the mouse brain induced convulsion and tremors at a smaller dose after a shorter latent period than that in systemic administration. The symptoms were prevented by pyridoxine, aminooxyacetic acid or acetone, while they enhanced by pyridoxal, pyridoxal phosphate, or some other anti-B6. In mice fed a vitamin B6 (B6)-deficient diet, convulsion and tremors occurred at smaller doses of SC than those in mice given control food, and were counteracted by pyridoxine. On the other hand, mice into which SC had been injected in the neighboring site of the lambda first showed running fits, which was followed by convulsion and tremors.
Simultaneous determination of vitamin D2 and its isomers, with the aid of pmr employing a paramagnetic shift reagent Eu (dpm)3, is presented. Thus, a mixture of vitamin D2 isomers is acetylated and its pmr spectra are run with and without the addition of the shift reagent. Relative paramagnetic shift values (νrel) of the acetoxyl protons of the acetylated isomers are estimated for solutions in CCl4 against the reference acetoxyl signal of vitamin D2 acetate. Estimated values are as follows: vitamin D2 acetate=100, 5, 6-traps-vitamin D2 acetate=71±1, ergosteryl acetate=86±1, lumisteryl2 acetate=123±3, isovitamin D2 acetate=82, isotachysteryl2 acetate=97, tachysteryl2 acetate=100 (105±1 in C6D6), and previtamin D2 acetate=80±2. On the basis of a dramatic shift of an acetoxyl signal and a constancy of each vrel value, this method permits immediate identification of the isomers and provides valuable information on their stereochemistry. This technique also facilitates a simultaneous determination of these isomers in a mixture by estimating each signal area.
The simultaneous determination of vitamin D2 and its isomers by high-speed liquid chromatography (HSLC) is described. Preferred operating conditions for the resolution and quantitation of vitamin D2, 5, 6-traps-vitamin D2, ergosterol, lumisterol2, isovitamin D2, isotachysterol2, tachysterol2, and previtamin D2 are as follows: column, “Zorbax” SIL (25cm×2.1mm i.d.); pressure, 100-120kg/cm2; temperature, ambient; detector, UV 254nm; mobile phase, 0.15% methanol+2% ether in pentane (for the simultaneous determination), 10% ether in hexane (for quantitation of the practical sets of isomers), or 55% CHCl3 (distilled) in pentane (for separation of isovitamin D2 and 5, 6-traps-vitamin D2); sample size, 1 μl (>1-5 ng); internal standard, p-cresol or α-naphthol. High-speed, good resolution, precise and accurate trace analysis, greater analysis flexibility, no necessity for preparing derivatives, and gentle operating conditions are main potential advantages of our method which has proved to be very efficient and surpasses all analytical procedures hitherto proposed.
The development of alkaline phosphatase influenced by 1α-OH-D3 (a synthetic active form of vitamin D3) and cortisone was studied in chick duodenal organ cultures. The administration of cortisone to the embryo in ovo on the 14th day of incubation resulted in a precocious increase in alkaline phosphatase after 6 days (20-day embryo). When duodena from 14-, 18- and 20-day embryos were cultured in the presence of cortisone, there was no significant enhancement of alkaline phosphatase activity except for a marginal effect observed in the 18-day duodenum. On the other hand, the alkaline phosphatase activity in cultured duodena from 20-day chick embryos was significantly stimulated by the addition of 1α-OH-D3. The effects of cortisone and 1α-OH-D3 were not additive. The activity of maltase, another intestinal enzyme, was not influenced by 1α-OH-D3. Studies on inactivation of alkaline phosphatase by EDTA suggest that the observed increase in alkaline phosphatase activity induced by the administration of 1α-OH-D3 in vitro may be related to the qualitative changes in the enzyme that take place during development in vivo.
Pharmacological studies were performed on allithiamine (TAD), thiamine propyl disulfide (TPD), and thiamine tetrahydrofurfuryl disulfide (TTFD) to investigate positive inotropic and negative chronotropic effects seen when they are applied to spontaneous beats in isolated guinea pig atria at concentrations higher than 10g/ml. 1. The effects of thiamine 8-(methyl-6-acetyldihydrothioacetate) disulfide (TATD) and thiamine hydroxyethyl disulfide (TOED) at 5×10-4 were slight, and those of dibenzoyl thiamine (DBT) and thiamine were limited at any concentration. 2. Dimethyl propyl disulfide (DMPD), which has anti-thiamine activity, showed these effects at concentrations higher than 10-5. 3. The negative chronotropic effect of TAD, TPD, and TTFD was not influenced by the prior application of atropine, and the positive inotropic effect was not influenced by propranolol. 4. The effects of TTFD on the electrically driven left atrial muscle were remarkable when the muscle was driven at low frequency, while they were less remarkable at high frequency. 5. The decrease in tension of the electrically driven left atria induced by mersalyl at 5×10-4g/ml was recovered by the subsequent addition of TTFD or TPD at 5×10-4 as well as dimercaprol at 5×10. From the results, it was assumed that (a) the effects of TAD, TPD, and TTFD might relate to their common chemical structure of a disulfide, especially to the alkyldisulfide chain, (b) the effects are irrelevant to their common activity as an vitamin B1 and to either cholinergic or adrenergic effect, and (c) a mutual dependence is seen between the positive inotropic effect and the negative chronotropic effect.
1. The spontaneous contractions of isolated guinea pig atria were arrested by a temperature change in the medium from 30 to 20°C within 20 min. 2. When thiamine tetrahydrofurfuryl disulfide (TTFD) at concentration of 10-5g/ml was added in the medium, the arrest was not seen for more than 30 min. 3. Arrhythmic contractions induced by the electrical square wave stimulation at threshold intensity were prevented by TTFD at 10-4g/ml which was added to the medium. 4. These effects of TTFD at respectivee concentrations were seen even 30 min after the drug was removed from the medium, when the atria had been pre-incubated with the drug for one hour before the removal. 5. From these results, it was assumed that TTFD might show these effects against the extrinsic physical invasions through the stabilization of the tissue membrane.
Gas-liquid chromatographic (GLC) determination of vitamin D in multivitamin preparations was investigated and a simple routine method was established. The unsaponifiable matters of a sample was applied to a thin-layer chromatography (TLC) plate using Kieselgel GF254 as an adsorbent and a mixture of n-hexane-ethyl acetate (4:1) as a developing solvent. The scraped zones corresponding to vitamin D and previtamin D were extracted and determined by GLC using 1.5 OV-17 packed on Shimalite w (80-100 mesh) as a stationary phase. When the proposed method was applied to model preparations made by mixing vitamin D, A and E, it was confirmed that the determination of vitamin D was possible when the ratios of vitamin A and E (DL-α-tocopheryl acetate) to vitamin D were within 104 (I. U, ratio) and 2, 500 (weight ratio), respectively. Since the ratios of most of commercial multivitamin preparations on sale in Japan are within the limitations and the results on multivitamin preparations were also satisfactory, the proposed method was confirmed to be suitable simple routine.
The effect of some reductones on glyoxalase I prepared from animal and microbial origins has been studied. The enzyme was extracted from ox liver or baker's yeast and partially purified by ammonium sulfate fractionation, gel filtration and ion exchange chromatography. Aliphatic reductones such as ascorbic acid, ascorbic acid 3phosphate and triose reductone showed strong to medium inhibition, while dehydroascorbic acid showed no inhibition. Kinetic analysis indicated that the inhibition mechanism of ascorbic acid was uncompetitive. Varying extents of inhibition were observed among three kinds of diphenols belonging to aromatic reductones. They were in the order of increasing inhibitory power resorcinol, hydroquinone and catechol for the ox liver enzyme, and catechol, resorcinol and hydroquinone for the yeast enzyme. p-Benzoquinone, an oxidized reductone, exhibited marked inhibition on both enzymes. Its action seemed due to reaction with amino and/or sulf hydryl functions of enzyme protein and those of glutathione, one of the substrates.