Akio Fujisawa

An unusual vitamin E constituent (alpha-tocomonoenol) provides enhanced antioxidant protection in marine organisms adapted to cold-water environments.

A new vitamin E constituent having an unusual methylene unsaturation at the isoprenoid-chain terminus of α-tocopherol (α-Toc) was isolated from chum salmon eggs and was found to have identical antioxidant activity as does α-Toc in methanol or liposomal suspension at 37°C. Here we report that this marine-derived tocopherol (MDT) is broadly distributed with α-Toc in the tissue of marine fish, and that the MDT composition of total vitamin E is greater in the flesh of cold-water salmon (12–20%) than in that of tropical fish (≤2.5%). Vitamin E analysis of cultured masu salmon maintained on a MDT-deplete diet showed substantially less MDT content than native masu salmon, suggesting a trophic origin of MDT. This contention is supported by the finding of MDT in marine plankton from the cold waters of Hokkaido. We found that MDT inhibited peroxidation of cholesterol-containing phosphatidylcholine liposomes to a greater extent than did α-Toc at 0°C. Furthermore, the ratios of the rate constants for MDT and α-Toc to scavenge peroxyl radicals increased with decreasing rates of radical flux in liposomes and fish oil at 0°C, indicating that the enhanced activity of MDT at low temperature is attributed to its greater rate of diffusion in viscous lipids. These results suggest that MDT production, or its trophic accumulation, may reduce lipid peroxidation in marine organisms functionally adapted to cold-water environments.

Repeated edaravone treatment reduces oxidative cell damage in rat brain induced by middle cerebral artery occlusion.

Abstract The free radical scavenger 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone) has been used to treat acute brain infarction in Japan since 2001. To obtain direct evidence that edaravone serves as an antioxidant in vivo, four groups of rats were prepared: (i) an ischemia/reperfusion (I/R) group receiving 2 h occlusion-reperfusion of the middle cerebral artery; (ii) a single administration group treated by intravenous infusion of edaravone (3 mg/kg) immediately after I/R; (iii) a repeated treatment group receiving twice daily edaravone administration for 14 days; and (iv) a sham operation group without occlusion. Repeated treatment with edaravone significantly improved the neurological symptoms and impairment of motor function as compared to the I/R group, while single administration demonstrated limited efficacy. No significant differences in plasma antioxidants such as ascorbate, urate, and vitamin E, or in redox status of coenzyme Q9 were observed among the four groups. In contrast, the plasma content of oleic acid in the total free fatty acids (percentage 18:1) was significantly increased in the I/R group for 7 days as compared to the sham operation group. Oleic acid was produced from stearic acid by the action of stearoyl-CoA desaturase to compensate for the oxidative loss of polyunsaturated fatty acids. The above results suggest that cellular oxidative damage in the rat brain is evident for at least 7 days after I/R. Repeated treatment suppressed the percentage 18:1 increment, while the single administration did not, which is consistent with the limited efficacy of single administration.

Saposin B Is a Human Coenzyme Q10-Binding/Transfer Protein

Coenzyme Q10 (CoQ10) is essential for ATP production in the mitochondria, and is an important antioxidant in every biomembrane and lipoprotein. Due to its hydrophobicity, a binding and transfer protein for CoQ10 is plausible, but none have yet been isolated and characterized. Here we purified a CoQ10-binding protein from human urine and identified it to be saposin B, a housekeeping protein necessary for sphingolipid hydrolysis in lysosomes. We confirmed that cellular saposin B binds CoQ10 in human sperm and the hepatoma cell line HepG2 by using saposin B monoclonal antibody. The molar ratios of CoQ10 to saposin B were estimated to be 0.22 in urine, 0.003 in HepG2, and 0.12 in sperm. We then confirmed that aqueous saposin B extracts CoQ10 from hexane to form a saposin B-CoQ10 complex. Lipid binding affinity to saposin B decreased in the following order: CoQ10>CoQ9>CoQ7>>α-tocopherol>>cholesterol (no binding). The CoQ10-binding affinity to saposin B increased with pH, with maximal binding seen at pH 7.4. On the other hand, the CoQ10-donating activity of the saposin B-CoQ10 complex to erythrocyte ghost membranes increased with decreasing pH. These results suggest that saposin B binds and transports CoQ10 in human cells.

Oxidative deterioration of platinum nanoparticle and its prevention by palladium

Abstract:  Pt nanoparticle is a strong reductant and has been used as an antioxidant in cosmetics and medicine. It was reported to have catalase‐like activity, which converts hydrogen peroxide to water and oxygen. However, in this study, freshly prepared Pt nanoparticle was almost inert towards decomposing hydrogen peroxide. The catalase‐like activity of Pt nanoparticle increased with increasing weeks of storage at room temperature and became more significant when the Pt nanoparticle was exposed to air. No hydroxyl radical formation was confirmed by several methods such as ESR spin‐trapping, dimethyl sulphoxide oxidation, salicylic acid hydroxylation and hydroxytoluene oxidation, indicating that the decomposition of hydrogen peroxide proceeds by the two‐electron oxidation/reduction reaction. The oxidatively deteriorated Pt nanoparticle catalytically decomposed ascorbic acid, which is one of the most important biological antioxidants. We found that such oxidation was effectively prevented by the addition of Pd nanoparticle. We also discussed the reaction mechanisms and application of Pt nanoparticle.

Edaravone, a potent free radical scavenger, reacts with peroxynitrite to produce predominantly 4-NO-edaravone

Objectives: 3-Methyl-1-phenyl-2-pyrazolin-5-one (edaravone) is used in clinical treatment of acute brain infarction to rescue the penumbra, based on its ability to prevent lipid peroxidation by scavenging lipid peroxyl radicals. Here, we show that edaravone also reacts with peroxynitrite to yield 4-NO-edaravone as the major product and 4-NO2-edaravone as a minor product. Results: We observed little formation of 3-methyl-1-phenyl-2-pyrazolin-4,5-dione (4-oxoedaravone) and its hydrate, 2-oxo-3-(phenylhydrazono)butanoic acid, which are the major free radical-induced oxidation products of edaravone, suggesting that free radicals are not involved in the reaction with peroxynitrite. The reaction of peroxynitrite with edaravone is approximately 30-fold greater than with uric acid, a physiological peroxynitrite scavenger (reaction rate k = 1.5 × 104 M−1 s−1 vs. 480 M−1 s−1). Discussion: These results suggest that edaravone functions therapeutically as a scavenger of peroxynitrite as well as lipid peroxyl radicals, which is consistent with a report that edaravone treatment reduced levels of 3-nitrotyrosine in the cerebrospinal fluid of patients with amyotrophic lateral sclerosis.

Coenzyme Q10-Binding/Transfer Protein Saposin B also Binds γ-Tocopherol

γ-Tocopherol, the major form of dietary vitamin E, is absorbed in the intestine and is secreted in chylomicrons, which are then transferred to liver lysosomes. Most γ-tocopherol is transferred to liver microsomes and is catabolized by cytochrome p450. Due to the hydrophobicity of γ-tocopherol, a binding and transfer protein is plausible, but none have yet been isolated and characterized. We recently found that a ubiquitous cytosolic protein, saposin B, binds and transfers coenzyme Q10 (CoQ10), which is an essential factor for ATP production and an important antioxidant. Here, we report that saposin B also binds γ-tocopherol, but not α-tocopherol, as efficiently as CoQ10 at pH 7.4. At acidic pH, saposin B binds γ-tocopherol preferentially to CoQ10 and α-tocopherol. Furthermore, we confirmed that saposin B selectively binds γ-tocopherol instead of CoQ10 and α-tocopherol at every pH between 5.4 and 8.0 when all three lipids are competing for binding. We detected γ-tocopherol in human saposin B monoclonal antibody-induced immunoprecipitates from human urine, although the amount of γ-tocopherol was much smaller than that of CoQ10. These results suggest that saposin B binds and transports γ-tocopherol in human cells.

Synergistic inhibition of lipid peroxidation by vitamin E and a dopamine agonist, cabergoline

Abstract We examined antioxidant activity of cabergoline, a dopamine agonist, during the aerobic oxidation of phosphatidylcholine liposomes at 37°C. Cabergoline retarded the oxidation initiated with a lipid-soluble initiator significantly better than that with a water-soluble initiator, suggesting that cabergoline locates in the lipid layer of liposomal membranes. Cabergoline inhibited the oxidation of liposomal membranes synergistically with endogenous antioxidants such as ascorbic acid, ubiquinol-10 and vitamin E, and vitamin E was the most efficient synergist. These results suggest that cabergoline may have a neuroprotective effect on the substantia nigra of Parkinsonian patients because of its synergistic antioxidant activity with vitamin E as well as its action on dopamine receptor.

Parabanic acid is the singlet oxygen specific oxidation product of uric acid

Uric acid quenches singlet oxygen physically or reacts with it, but the oxidation product has not been previously characterized. The present study determined that the product is parabanic acid, which was confirmed by LC/TOFMS analysis. Parabanic acid was stable at acidic pH (<5.0), but hydrolyzed to oxaluric acid at neutral or alkaline pH. The total yields of parabanic acid and oxaluric acid based on consumed uric acid were ~100% in clean singlet oxygen production systems such as UVA irradiation of Rose Bengal and thermal decomposition of 3-(1,4-dihydro-1,4-epidioxy-4-methyl-1-naphthyl)propionic acid. However, the ratio of the amount of uric acid consumed to the total amount of singlet oxygen generated was less than 1/180, indicating that most of the singlet oxygen was physically quenched. The total yields of parabanic acid and oxaluric acid were high in the uric acid oxidation systems with hydrogen peroxide plus hypochlorite or peroxynitrite. They became less than a few percent in peroxyl radical-, hypochlorite- or peroxynitrite-induced oxidation of uric acid. These results suggest that parabanic acid could be an in vivo probe of singlet oxygen formation because of the wide distribution of uric acid in human tissues and extracellular spaces. In fact, sunlight exposure significantly increased human skin levels of parabanic acid.

Stabilizers of edaravone aqueous solution and their action mechanisms. 1. Sodium bisulfite

Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) has been used as a free radical scavenging drug for the treatment of acute ischemic stroke in Japan since 2001. Edaravone is given to patients intravenously; therefore, it is distributed in the form of an aqueous solution. However, aqueous solutions of edaravone are very unstable because it is present as edaravone anion, which is capable of transferring an electron to free radicals including oxygen, and becomes edaravone radical. We observed the formation of hydrogen peroxide and edaravone trimer when aqueous edaravone solution was kept at 60°C for 4 weeks. We proposed the mechanism of edaravone trimer formation from edaravone radicals. Lowering the pH and deoxygenation can effectively increase the stability of aqueous edaravone solution, since the former reduces edaravone anion concentration and the latter inhibits edaravone radical formation. Addition of sodium bisulfite partially stabilized aqueous edaravone solutions and partially inhibited the formation of edaravone trimer. Formation of bisulfite adduct was suggested by 13C NMR and HPLC studies. Therefore, the stabilizing effect of sodium bisulfite is ascribed to the formation of a bisulfite adduct of edaravone and, consequently, reduction in the concentration of edaravone anion.

5- N -Carboxyimino-6- N -chloroaminopyrimidine-2,4(3 H )-dione as a hypochlorite-specific oxidation product of uric acid

Although uric acid is known to react with many reactive oxygen species, its specific oxidation products have not been fully characterized. We now report that 5-N-carboxyimino-6-N-chloroaminopyrimidine-2,4(3H)-dione (CCPD) is a hypochlorite (ClO−)-specific oxidation product of uric acid. The yield of CCPD was 40–70% regardless of the rate of mixing of ClO− with uric acid. A previously reported product, allantoin (AL), was a minor product. Its yield (0–20%) decreased with decreasing rate of mixing of ClO− with uric acid, indicating that allantoin is less important in vivo. Kinetic studies revealed that the formation of CCPD required two molecules of ClO− per uric acid reacted. The identity of CCPD was determined from its molecular formula (C5H3ClN4O4) measured by LC/time-of-flight mass spectrometry and a plausible reaction mechanism. This assumption was verified by the fact that all mass fragments (m/z −173, −138, −113, and −110) fit with the chemical structure of CCPD and its tautomers. Isolated CCPD was stable at pH 6.0–8.0 at 37°C for at least 6 h. The above results and the fact that uric acid is widely distributed in the human body at relatively high concentrations indicate that CCPD is a good marker of ClO− generation in vivo.