The effects of administration of DL-pemcillamine (PeA), thiosemicarbazide (TSC), semicarbazide-HCl (SC) as convulsants and pyridoxine (PN) as anticonvulsant on γ-aminobutyric acid (GABA) content, glutamic acid decarboxylase (GAD) and γ-aminobutyric acid transaminase (GABA-T) activities in cerebral cortex, striatum, dien-cephalon, mesencephalon, cerebellum and pons/medulla were investi-gated. The onset of convulsions induced by these convulsants coincides with the fall in GABA content and GAD activity in the mesencephalon area, and in contrast, the cessation of the convulsions by PN supplement coincides with the recovery in both the parameters. Aminooxyacetic acid (AOAA), a potent GABA-elevating agent showed an anticonvulsant property against convulsion by TSC for several hours after the injection of AOAA, but lost this property 16hr after the treatment. The TSC administration 16hr after the AOAA pretreatment significantly decreased the GABA content in all the regions, particularly in the mesencephalon and diencephalon areas, which had been elevated by the AOAA pretreatment, together with its ability to induce convulsion. From the above results it may be postulated that the critical drop of GABA level from a plateau to another lower level following the decrease of GAD activity in the mesencephalon area is an important factor in the induction of convulsion.

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A method using preparative and analytical high-performance liquid chromatography (HPLC) is proposed for the assay of 25-hydroxyvitamin D₃ (25-OH-D₃) in human plasma. A constant volume (0.2-2.0ml) of a plasma sample was saponified. The unsaponifiable matter was first applied to preparative HPLC using a column of the straight-phase type (Zorbax SIL) in order to separate a 25-OH-D₃ fraction from lipophilic concomitants giving ultraviolet-absorbing noise. Then, the separated 25-OH-D₃ faction was applied to analytical HPLC using a column of the reversed-phase type (Zorbax ODS) in order to measure the content of 25-OH-D₃ from the peak height. This is a revised method from JONES ((1978): Gun. Chem., 24, 287-298). The results showed that the clean-up procedure by the first preparative HPLC was successfully performed because the peak corresponding to 25-OH-D₃ on the chromatogram of the second analytical HPLC was not disturbed by any other interfering peaks. Moreover, recovery through the whole procedure was satisfactory (about 100%) and the procedures of saponification and isolation of the unsaponifiable matter diminished the overload to the columns. These are the revised points of Jones' method. When two determinations were performed on 12 samples of plasma taken from normal adults in October, the values were 22.6±4.8 and 21.0±3.6 (mean±SD) ng/ml, respectively.

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In order to obtain a standard compound of 25-hy-droxyvitamin D₂ (25-OH-D₂), a method for isolating in vivo-generated 25-OH-D₂ from the blood of rats or rabbits was established by using several steps of preparative high-performance liquid chromatography (HPLC). When the unsaponifiable matter of the plasma obtained from rats or rabbits receiving a large dose of vitamin D₂ was applied to the preparative HPLC using a Zorbax SIL column, a peak denoted as peak X was observed on the chromatogram. Since the peak X was thought to be due to 25-OH-D₂ from the experiments of time course and dose-response, it was purified by subjecting it to successive preparative HPLC using several kinds of columns. From the results of ultraviolet (UV) absorption spectrum, gas chromatography-mass spectrometry (GC-MS) and mass chromatography, the purified peak X compound was confirmed to be 25-OH-D₂. The proposed method for isolating in vivo-generated 25-OH-D₂ is very convenient, because the time to perform each HPLC is very short though several steps of HPLC are used.

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The binding of metabolically activated ¹⁴C-benzo(a)pyrene (BP) to rat liver nuclei was studied. The nuclear framework structure, termed the nuclear matrix, was found to bind BP preferably. α-Tocopherol inhibited the binding of BP to nuclear macromolecules in the presence of phenobarbital (PB)-induced microsomes. α-Tocopherol decreased the level of BP metabolites in nuclei, though it did not inhibit the activation of BP by PB-induced microsomes.

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The study was undertaken on alpha-tocopherol levels, separated from other analogs, in the red blood cells (RBC) of cord blood, premature infants, healthy children and adults as well as in pregnant women, and compared with the plasma levels.
1. The majority of tocopherol found in the RBC was localized in the membranes.
2. Only alpha-tocopherol was found in the RBC, while alpha- and gamma-tocopherol were found in the plasma.
3. Alpha-tocopherol level in the RBC changes during development in parallel with that in the plasma. However, changes in the level in RBC were smaller than those of the plasma.
4. The ratio of alpha-tocopherol level in the RBC to the plasma was higher in the cord blood and premature infants than in the children and adults.
5. With regard to the cases with high plasma tocopherol levels, a different finding was obtained on the RBC level between pregnant women and adults with a large amount of tocopherol. In the latter, high RBC levels were observed as the plasma levels were elevated, while in spite of a high plasma tocopherol level, the lowest RBC levels were found in the pregnant women.

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Thiamine and its phosphate esters in rat tissues were determined by high-performance liquid chromatography after conversion to thiochrome and its phosphate esters as described previously (Ishii, K. et al. (1979) Anal. Blochem., 97, 191-195). Total thiamine concentrations determined in the brain, liver, heart and kidney were 6.39, 25.2, 15.7, and 14.9 nmol/g wet weight, respectively. In all the tissues tested, thiamine, its monophosphate, pyrophosphate, and triphosphate were found to be present at the rates of 1.2-7.7, 5.9-11.0, 85.7-90.0, and 0.7-1.6%, respectively. Although these percentages of thiamine phosphates were similar to those reported previously using different procedures, the percentage of thiamine triphosphate content in rat tissues was found to be fairly lower than that reported. The time required for the whole process of analysis averaged 90min after isolation of the organs from the animal.

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The present study examined lipid transport from the liver by application of a perfusion technique in situ in rats fed diets consisting of a low level Wheat- or Rice- or Miyazaki-pattern amino acid mixture. The net release of hepatic lipids was directly estimated from analysis of the perfusate continuously circulated through the liver. The net release of triacylglycerol from the liver into the perfusate was much more severely inhibited in the Wheat-pattern diet group, and more depressed in the Rice-pattern diet group than in the control group, and slightly reduced in the Miyazaki-pattern diet group compared with the control. For serum lipoproteins fractionated by the Quick-Disc electrophoresis method, there was a significant decrease in the serum pre-β lipoprotein fraction in all the rats of the Wheat-, Rice- and Miyazaki-pattern diet groups compared with the control. These observations provide firm support for the hypothesis that in rats given diets consisting of a low level of the Wheat-, Rice- and Miyazaki-pattern amino acid mixture, fatty liver may be produced mainly by an impaired transport of triacylglycerols from the liver.

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Characteristics of early appearing free isomaltase in the soluble fraction were investigated in rat intestinal mucosa. Soluble isomaltase and membrane-bound sucrase-isomaltase complex were pre-pared from 15-day-old rat intestine. Immunochemical properties, optimal pH and heat sensitivity of soluble isomaltase were compared with those of membrane-bound isomaltase.
Optimal pH of free isomaltase in the soluble fraction was lower than that of membrane-bound isomaltase. Soluble and membrane-bound isomalt-ase showed different sensitivities for temperature. Furthermore, membrane-bound isomaltase in 15-day-old suckling rat intestine gave a single line with antiserum. However, soluble isomaltase gave no precipitin line. From these results, it could be concluded that soluble isomaltase is not derived from the isomaltase moiety of membrane-bound sucrase-isomaltase complex as a result of mechanical fragility and rather it would be lysosomal in origin.

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