Hiroshi Yasuda

Biphasic Liberation of Arachidonic and Stearic Acids During Cerebral Ischemia

Abstract: The mode of free fatty acid (FFA) liberation from the mouse brain during ischemia was investigated at various times after decapitation and under nizofenone treatment. Normal nonischemic brain FFAs consist mainly of palmitic acid (16:0), stearic acid (18:0), and oleic acid (18:1) with smaller amounts of arachidonic acid (20:4), docosahexaenoic acid (22:6), and others. Postde‐capitative ischemia induced a rapid, biphasic release of 20:4 after a short lag of <30 s. The first phase showed a rapid 6.4‐fold increase within 1 min of decapitation, followed by the second phase involving a slow release at less than one‐fifth the rate of the first phase and lasting for at least 10 min. A similar, but not so marked, biphasic liberation was observed with 18:0. However, all of the other fatty acids (16:0, 18:1, 22:6, and others) were released only in a single phase at a slow rate. The time course for the rapid and specific liberation of 20:4 coincided with the time course for the decrease in brain ATP concentration during ischemia. Pretreatment of the animals with nizofenone resulted in a marked suppression of both FFA liberation and ATP depletion during ischemia. This suppression was particularly noteworthy with 20:4 and 18:0. The present study indicates that there is a specific and rapid liberation of 20:4 and 18:0 in a very early stage of ischemia and that this liberation seems to depend on availability of ATP in the brain. The physiological role of this transient 20:4 liberation during ischemia is discussed.

Mechanism of the Salicylate hydroxylase reaction': II. The enzyme-substrate complex

Abstract 1. 1. Salicylate hydroxylase (salicylate, NADH:oxygen oxidoreductase (1-hydroxylating, 1-decarboxylating)) from Pseudomonas putida forms an enzyme-substrate complex with salicylate. 2. 2. The complex could be detected by a new absorption maximum around 480 nm. By spectrophotometric titration, it was found that a molar ratio of apoenzyme, FAD and salicylate in the complex was I:I:I. 3. 3. The complex was more stable than the holoenzyme under any tested conditions, i.e. , heat, acid and proteinase treatments. 4. 4. The FAD moiety of the complex was reduced with NADH under anaerobic conditions, and the reoxidation of the reduced complex with air resulted in product formation. The stoichiometric relation in each reaction was demonstrated by using substrate level amounts of the enzyme. A mechanism for salicylate hydroxylation reaction is proposed.

Cerebral Protective Effect and Radical Scavenging Action

Abstract The role of radical scavenging action in cerebral protective effect of drugs was investigated in vitro. Incubation of rat brain mitochondrial suspension with ascorbic acid and Fe2+ resulted in the formation of malondialdehyde and a decrease in the turbidity of the suspension, indicating that the mitochondria were peroxidatively disintegrated. Nizofenone at 10 μm or more inhibited the peroxidative disintegration of mitochondria, and complete inhibition was observed at 100–200 μm. The action of nizofenone was also ascertained by experiments with rat liver mitochondria. The anti‐peroxidative activity of nizofenone was estimated to be approximately equivalent to that of α‐tocopherol, and this property was unique. Among the cerebral protective drugs tested, thiopental was only slightly efficient, and pentobarbital, phenobarbital, and dimethyl sulfoxide had no effect. In addition, nizofenone was found to scavenge a stable free radical, diphenyl‐p‐picrylhydrazyl, but the barbiturates did not. These findings suggest that there is no intimate relationship between cerebral protective effect and free radical scavenging action.

Lipid peroxidation and its inhibition by tinoridine. I. Lipid peroxidation-induced disintegration of microsomal membrane and cytochrome P-450 in rat liver.

Abstract Incubation of rat liver microsomes with ascorbic acid resulted in the formation of malondialdehyde, the destruction of cytochrome P-450 and the decrease in the turbidity of the microsomal suspension. Tinoridine (2-amino-3-ethoxy-carbonyl-6-benzyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine), at the concentration of 5 μM, completely inhibited the peroxidative damages of microsomes. A similar inhibition by tinoridine was observed in NADPH-induced lipid peroxidation of microsomes. The anti-peroxidative activity of tinoridine was about 50 times greater than that of α-tocopherol. From the relationship between the concentration of tinoridine and the amount of malondialdehyde formed, it was demonstrated that 1 mol of tinoridine blocks the formation of about 10 mol of malondialdehyde. This suggests that lipid peroxidation proceeds as a chain reaction and that tinoridine inhibits the reaction at the initial step.

Mechanism of the salicylate hydroxylase reaction: III. Characterization and reactivity of chemically or photochemically reduced enzyme-flavin

Abstract 1. 1.|The spectral natures of chemically- and photochemically-reduced salicylate hydroxylase (salicylate, NADH: oxygen oxidoreductase (1-hydroxylating, 1-decarboxylating)) from Pseudomonas putida have been investigated. 2. 2.|When salicylate hydroxylase in the presence of substrate was titrated with a limited amount of dithionite or was illuminated in the presence of EDTA, a new species with a weak absorption band at a long wavelength appeared, whereas an entirely different species which was typical for the “red flavoprotein radical” was detected in the absence of substrate. The latter was converted to the former upon anaerobic addition of substrate. 3. 3.|Reoxidation of the reduced enzyme-substrate complex with air formed the product in an amount stoichiometric with the reduced flavin.

A reliable analysis of tissue free fatty acids by gas-liquid chromatography

A convenient and reliable gas-liquid chromatographic method for determining the free fatty acids in biological specimens is described. The free fatty acids were extracted with hexane in the presence of H3PO4 and then back-extracted from the hexane phase into a very small volume of trimethyl (alpha, alpha, alpha-trifluoro-m-tolyl)ammonium hydroxide solution. Direct injection of the resultant quaternary ammonium salts of the fatty acids into a gas-liquid chromatograph unit gave their methyl esters, with a high recovery. The presence of triglycerides, phospholipids, or cholesterol esters did not interfere with the determination of free fatty acids. This method was applied to determination of free fatty acids in the samples of serum or brain. The results were more precise and reliable than those reported with the conventional methods with TLC separation. This method should be a useful aid for providing precise information about the physiological or pathological roles of free fatty acids.

Hydroxyl radical scavenging action of tinoridine

Radical scavening action of tinoridine, a non-steroidal anti-inflammatory drug with a potent antiperoxidative activity, was investigated. Tinoridine reduced a stable free radical, diphenyl-p-picrylhydrazyl, in the molar ratio of about 1∶2, indicating its free radical scavenging ability. Tinoridine inhibited the lipid peroxidation in rat liver microsomes induced by xanthine-xanthine oxidase system in the presence of ADP and Fe2+, in which hydroxyl radical (·OH) is formed. Tinoridine was demonstrated to be oxidized in the course of the lipid peroxidation by following the fluorescence derived from the oxidation product of tinoridine. It was also oxidized by the xanthine-xanthine oxidase system in the presence of Fe2+, but its oxidation was slow in the absence of Fe2+ and almost completely inhibited by catalase. Tinoridine was also oxidized by H2O2−Fe2+ system producing ·OH (Fenton reaction), but it did not affect the reduction of cytochrome c caused by superoxide radical. These results indicate that tinoridine is able to scavenge ·OH and the main active oxygen species responsible for the lipid peroxidation is ·OH. The anti-peroxidative and ·OH scavenging ability of tinoridine should contribute to its anti-inflammatory action.

Y-8894の薬理学的研究(第4報)

The amelioration of energy metabolic disturbance in cerebral anoxia is valuable for the treatment of various cerebral ischemic diseases and insufficiency. In this study, the effect of Y-8894 on the cerebral energy metabolism was investigated using a KCN-induced cerebral anoxia model with mice. The intravenous injection of a lethal dose of KCN (2.5 mg/kg) induced rapid and marked decreases of brain glucose, phosphocreatine and ATP contents, with a remarkable enhancement of lactate and AMP levels, indicating a severe disorder of the cerebral energy metabolism. This phenomenon was also shown by an irreversible deterioration of the energy charge potential (ECP), an index of the cerebral energy state. The treatment with Y-8894 (30 mg/kg, i.p.) remarkably ameliorated this KCN-induced energy metabolic disturbance: markedly reducing the changes in brain phosphocreatine, glucose and lactate contents, while keeping ATP, AMP and ECP at nearly their normal levels. In addition, these changes in the Y-8894 treated group recovered promptly to normal, whereas those in the control group were irreversible. In normal mice, Y-8894 induced a significant increase in the cerebral glucose content without affecting either the cerebral glycolytic metabolism or the energy state. The present findings suggest that Y-8894 has an ameliorative effect on the cerebral energy metabolic disturbance, and this effect likely plays an important role in the improvement of amnesia and other neurological deficits related to cerebral anoxia.

Protection against hepatic injury by a novel spiropiperidine derivative.

The evaluation of a novel hepatoprotective agent, Y-8845 (8(2-dimethylaminoethyl)-3-oxo-4-phenyl-1-thia-4,8-diazaspiro [4,5]decane dihydrochloride monohydrate), against carbon tetrachloride (CCl4)- and endotoxin-induced acute and chronic hepatic injury was carried out in rats. This compound, in a dose-dependent way, markedly reduced the increases in serum transaminase activities, the extent of liver cell necrosis, and the delay in indocyanine green (ICG) disappearance produced by a single toxic dosage of CCl4. This protective effect was observed even at doses of Y-8845 lower than 10 mg/kg po. It was also shown to protect the liver against injury induced by endotoxin. Furthermore, in the chronic liver injury induced by repeated administrations of CCl4 for 12 weeks, significant reductions of the increases in serum enzyme activities, liver fibrosis, and liver enlargement, and improvement in the ICG retention rate, were recognized in the Y-8845-treated groups at 10 mg/kg po or less. These findings indicate that this new agent has a remarkable protective effect, and possibly a therapeutic effect on liver injury.