Four triacylglyceride hydroperoxides were synthesized by DCC-mediated esterification of a dimethylperketal of 13-hydroperoxy-octadecadienoic acid with glycerides, in which one or two linoleoyl groups were linked, and by final removal of the protective group with a mixture of THF, acetic acid and water.

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  Synthetic phosphatidylcholine hydroperoxide (PC-), phosphatidylethanolamine hydroperoxide (PE-) and phosphatidylglycerol hydroperoxide (PG-) could be analyzed by ion spray ionization mass spectrometry, clearly affording the molecular ion peaks as protonated ion species without any fragmentation. Linearity between PC- concentration and peak intensity, and the minimum detectable concentration of PC- were obtained.

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Squalene (SQ), a main component of human sebum, is readily photooxidized by exposure to sunlight, producing six squalene monohydroperoxide (SQ-

) isomers. Despite its known connection to various skin conditions, few studies have sought to analyze SQ- at the isomeric level. In this study, we aimed to develop a method to discriminate each SQ- isomer with the use of tandem mass spectrometry (MS/MS). The six standard SQ- isomers were prepared by photooxidizing SQ in the presence of rose bengal, a photosensitizer, and isolated by semipreparative high-performance liquid chromatography (HPLC). To purify each isomer, 2-methoxypropene, which reversibly reacts with the hydroperoxide group of SQ-, was utilized. Product ion scanning was then performed on the standard SQ- isomers in the absence and presence of the sodium ion. In the absence of the sodium ion, the fragmentation patterns produced by atmospheric pressure chemical ionization were similar between the isomers, whereas in the presence of the sodium ion by electrospray ionization, unique fragmentation patterns were achieved. Based on these fragment ions, HPLC-MS/MS multiple reaction monitoring analysis was conducted on a mixture of the standard SQ- isomers. We achieved discrimination of SQ- isomers with high selectivity and detected SQ- isomers at nanogram levels. These results may improve our understanding of the effect of SQ- on skin conditions as well as the mechanism behind SQ peroxidation.

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We present a density functional theory calculation for the adsorption and dissociation of on Pt(111) and Pt(111)-alloy surfaces. We confirmed the theoretical understanding of an activated dissociation on Pt(111) surface and on small Pt clusters. Interestingly, in this work, we found an existence of a “barrierless” dissociation on several Pt-binary and ternary alloy surfaces with Ru and Mo as alloying components: PtRu and PtRuMo. Here, we demonstrate how such reaction proceeds and discuss the role of Ru—O and Mo—O in the spontaneous dissociation in these systems. The reaction energetics of specie is one of the most sought fundamental surface science studies due to its importance in many catalytic and surface reactions such as hydrogen fuel cell. [DOI: 10.1380/ejssnt.2011.352]

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Lipid peroxidation proceeds by a free radical chain reaction yielding lipid hydroperoxides (L-) as the major initial reaction product. Moreover, the unsaturated fatty acids that constitute a large component of a lipid emulsion, such as Intralipid^TM, are highly susceptible to peroxidation. To determine the degree of lipid peroxidation in artificial lipid emulsions, we measured the content of triacylglycerol hydroperoxides (TG-) and phospholipid hydroperoxides (PL-) in 10% Intralipid^TM solution. The chemiluminescence-based high-performance liquid chromatography (HPLC) assay is the one of the most advanced methods for the direct detection of L- at picomole levels. Using this method, TG- and PL- could be detected in Intralipid^TM (166.7±83.9μmol/l and 3.5±2.9μmol/l, respectively) . The concentration of TG- and PL- in Intralipid^TM was significantly increased (228.0±106.8μmol/l (p<0.01) and 30.4±14.3μmol/l (p<0.02), respectively), during a 24 h infusion under ambient light and room temperature (26°C) in NICU with a concomitant reduction of emulsion α-tocopherol to 97% (55.9±4.7 mmol/l, p<0.05) compared with baseline levels (57.3±4.3mM) . This observation suggests that endogenous α-tocopherol could not prevent the formation of L- in Intralipid^TM over a 24 h period. Thus, it is clear that lipid infusion has greater nutritional advantages for small, premature infants. Furthermore, we should consider that lipid emulsions are prone to peroxidation, and that this may be an important feature of oxidant-associated tissue damage.

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The comedogenicity of squalene peroxides was examined on the rabbit ear skin after topical application of squalene-monohydroperoxide (Sq-), the initial product when squalene was irradiated with UV-A. Since comedogenic products from UV-irradiated squalene were extracted with methanol solution, we isolated Sq- by reverse-phased HPLC with a methanol mobile phase solvent. The degree of comedogehnic reaction induced by Sq- was higher than that of well-known comedogenic cosmetic ingredients. Unlike two other mono-peroxides, tert-butyl hydroperoxide and cumene-mono-hydroperoxide, Sq- induced comedo-formation in the rabbit ear skin. However, the comedogenicity of reduced SqOOH, squalene-hydroxide (Sq-OH) and squalene itself was lower than that of Sq-. These results indicate that Sq- is a potent comedogenic mono-hydroperoxide chemical to rabbit skin.

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Levels of phosphatidylcholine hydroperoxide (PC-) in plasma, red blood cell (RBC), and livers of cultured and commercially available marine and freshwater fish were determined quantitatively by normal phase high-performance liquid chromatography (HPLC) equipped with a post-column detection system, using diphenyl-1-pyrenyl phosphine as a fluorescence reagent. The levels of plasma PC- in non-aromatic fish, including yellowtail, amberjack, the Japanese flounder, sea bream, and rainbow trout ranged between 1.2 and 5.1 nmol/mL plasma. In contrast, the levels of PC- in the plasma of aromatic fish, including the Japanese smelt, rainbow smelt, shishamo smelt, and sweet smelt were extremely high. The sweet smelt plasma contained the highest amount of hydroperoxide, amounting to 29.4 nmol/mL plasma. The levels of PC- in non-aromatic fish RBC were low, ranging between 23.4 and 25.2 fmol/10⁵ RBC. In contrast, a large amount of PC- accumulated in all of the aromatic fish RBC used in the present study, ranging between 122 and 419 fmol/10⁵ RBC. The PC- levels in the sweet smelt and the Japanese smelt RBC were markedly high. The sweet smelt liver also contained a large amount of PC-, amounting to 670 nmol/g tissue. This value was five times higher than those of some non-aromatic fish. Any marked difference in the contents of phospholipids and polyunsaturated fatty acids in the blood of sweet smelt and rainbow trout was not recognized. These results suggest that biogeneration of lipid hydroperoxide is an initial step in the development of certain volatile compounds in the aromatic fish.

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An ethyl-labeled phosphatidylcholine hydroperoxide (PC-/Et 2) was synthesized as a molecular probe for naturally occurring PC- 1. Applying the precursor ion scan mode in tandem ESI mass spectrometry at m⁄z 198, a signal of the PC-/Et 2 alone could be selectively detected even in the presence of a large excess of a complex mixture of phospholipids in the blood. Furthermore, molecular species that formed from PC-/Et 2 by its degradation in the blood were also observed in the same spectrum. Since the molecular probe-and-mass spectrometry-assisted analytical method presented herein requires no separation process by HPLC or TLC and is speedy, requiring less than 1 h, it may be useful in lipid analysis.

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The oxidase reaction of lipoamide dehydrogenase with NADH generates superoxide radicals and hydrogen peroxide under aerobic conditions. ESR spin trapping using 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) was applied to characterize the oxygen radical species generated by lipoamide dehydrogenase and the mechanism of their generation. During the oxidase reaction of lipoamide dehydrogenase, DMPO- and DMPO-OH signals were observed. The DMPO- signal disappeared on addition of superoxide dismutase. These results demonstrate that the DMPO- adduct was produced from the superoxide radical generated by lipoamide dehydrogenase. In the presence of dimethyl sulfoxide, a DMPO-CH₃ signal appeared at the expense of the DMPO-OH signal, indicating that the DMPO-OH adduct was produced directly from the hydroxyl radical rather than by decomposition of the DMPO- adduct. The DMPO-OH signal decreased on addition of superoxide dismutase, catalase, or diethylenetriaminepentaacetic acid, indicating that the hydroxyl radical was generated via the metal-catalyzed Haber-Weiss reaction from the superoxide radical and hydrogen peroxide. Addition of ferritin to the NADH-lipoamide dehydrogenase system resulted in a decrease of the DMPO- signal, indicating that the superoxide radical interacted with ferritin iron.

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To elucidate further the mechanism of lipid hydroperoxide (LHP) induced neovascularization, we determined the effect of linoleic acid hydroperoxide (18:2-) on bovine aortic endothelial cells (BAEC) in terms of cell proliferation, migration, and tube formation. The influence of some antioxidants on these systems were also investigated. The concentration of basic fibroblast growth factor (bFGF) in the culture medium was determined by an immunoassay. Exposure to 10^-7M 18:2- increased BAEC proliferation, migration, and tube formation by 117, 167, and 181%, respectively, as compared with control values. The concentration of bFGF in the culture medium was increased 3 fold by 10^-7M 18:2- exposure for 3h, compared with that of controls (5.1 vs. 1.7pg/mg protein). BAEC migration induced by 10^-7M 18:2- was inhibited significantly by 10^-7M bucillamine (p<0.05), which contains two sulfhydryl groups; by 10^-7M troglitazone (p<0.05), which structurally similar to α-tocopherol; and by 10^-7M EPC-K1 (p<0.01), an α-tocopherol and ascorbic acid conjugate. Antioxidants showed marginal effects on proliferation. The de novo synthesis of bFGF after the 18:2- stimulus for 3h was reduced from 5.1pg/mg protein to 2.0pg/mg protein by treatment with bucillamine. These results suggest that 18:2- induced BAEC growth is partly related to bFGF release, or synthesis.

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By employing EPR spectrometry with the aid of a spin-trapping agent, 5, 5-dimethyl-l-pyrroline-l-oxide (DMPO), the generation of superoxide anion and hydroxyl radical was reevaluated during the respiratory burst of porcine and human neutrophils. Properly prepared resting neutrophils did not generate any spin-trapped radical, and, when the cells were stimulated with phorbol myristate acetate, only DMPO-, the spin-trapped adduct of superoxide anion, was detected. No formation of DMPO-OH, the spin-trapped adduct of the hydroxyl radical, was observed. DMPO- was also detected principally when the neutrophils were stimulated with opsonized zymosan, a particulate stimulus. In the latter case, however, the formation of DMPO- ceased shortly after the addition of zymosan and subsequent production of DMPO-OH was observed. The production of DMPO-OH was found to be associated with cell injury. DMPO at the concentration usually used for the experiment (0.045-0.09M) injured phagocytizing neutrophils, causing lysis of the cells. On the other hand, an addition of cell homogenate or glutathione-glutathione peroxidase system to the suspension of intact cells which were producing DMPO- resulted in the formation of DMPO-OH. Thus, DMPO-OH was probably derived from DMPO- by the action of enzymes and/or factor(s) which were released from the lysed cells.

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We have proposed that diacylglycerol hydroperoxide-induced unregulated signal transduction causes oxidative stress-related diseases. In this study, we investigated which molecular species of diacylglycerol hydroperoxide activated human peripheral neutrophils. All diacylglycerol hydroperoxides, diacylglycerol hydroxides, and diacyglycerols tested in the present study induced superoxide production by neutrophils. The ability to activate neutrophils among molecular species containing the same fatty acid composition was as follows; diacylglycerol hydroperoxide>diacylglycerol hydroxide≥diacylglycerol. The diacylglycerol hydroperoxide composed of linoleate was a stronger activator for neutrophils than that composed of arachidonate. 1-Palmitoyl-2-linoleoylglycerol hydroperoxide (PLG-) was the strongest stimulator for neutrophils. We reconfirmed that PLG- activated protein kinase C (PKC) in neutrophils. PLG- induced the phosphorylation of p47^phox, a substrate of PKC and a cytosolic component of NADPH oxidase, in neutrophils, as did N-formyl-methionyl-leucyl-phenylalanine or 4β-phorbol-12β-myristate-13α-acetate. Moreover, the time course of p47^phox phosphorylation was comparable to that of superoxide production. These results suggest that PLG- activated intracellular protein kinase C. PLG-, produced via an uncontrolled process, can act as a biological second messenger to cause inflammatory disease from oxidative stress.

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The oxidation of equivalent concentrations of phospholipids in homogeneous solution, in multilamellar liposomal suspension, and in rat liver homogenate was carried out under aerobic conditions at 37°C in order to examine the biochemical fate of oxidized phospholipids. Rat liver phospholipids were extracted with chloroform and methanol, and oxidation in this homogeneous solution was initiated with a lipid-soluble radical initiator. The oxidation products were phosphatidylcholine hydroperoxide (PC-) and phosphatidyl-ethanolamine hydroperoxide (PE-), which were quantified by HPLC separation using a hydroperoxide-specific chemiluminescence detector. Co-extracted a-tocopherol and ubiquinol-9 suppressed the formation of PC- and PE- until oxidatively exhausted. The oxidation of extracted rat liver phospholipids in multilamellar liposomal suspension initiated with the lipid-soluble initiator gave similar results, but with slower rates of antioxidant depletion and phospholipid hydroperoxide formation due to a lower efficiency of free radical production in liposomal membranes. In contrast, the oxidation of rat liver homogenate containing active tissue enzymes initiated by the addition of either free radical initiators or tert-butyl hydroperoxide gave phosphatidylcholine hydroxide, phospha-tidylethanolamine hydroxide, and free fatty acid hydroxides as oxidation products. Exogenous PC- added to the rat liver homogenate was reduced to phosphatidylcholine hydroxide with subsequent hydrolysis to its free fatty acid hydroxide. These results suggest that peroxidase and phospholipase enzymes play important roles in the repair of oxidatively damaged phospholipids in biomembranes.

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空気下37℃での水溶性および脂溶性のラジカル開始剤によるヒト血しょうの酸化におけるボスファチジルコリンヒドロペルオキシド (PC-), ボスファチジルコリンヒドロキシド (PC-OH), コレステロールエステルヒドロペルオキシド (CE-), コレステロールエステルヒドロキシド (CE-OH), 遊離脂肪酸ヒドロペルオキシド (FFA-), 遊離脂肪酸ヒドロキシド (FFA-OH) の生成および尿酸, アスコルビン酸, ビリルビン, ユビキノールー10, α-トコフェロールなどの酸化防止剤の変化を測定した。
水溶性開始剤による迅速なアスコルビン酸の減少と脂溶性開始剤による迅速なユビキノール-10とα-トコフェロールの減少は, 水溶性開始剤が水相で, 一方脂溶性開始剤は脂質相で作用していることを示唆する。
血しょうの酸化によりPC-とCE-に加えて相当量のPC-OHが生成した。PC-を血しょうに加えるとPC-OHが主として生成することから, PC-OHはペルオキシダーゼにより生成したと考えられる。
血しょうの酸化により相当量のCE-OHも生成した。血しょう中でCE-が比較的安定であることから, CE-OHはレシチン : コレステロールアシルトランスフェラーゼの作用によりPC-OHから生成したと考えられる。
FFA-とFFA-OHがほとんど生成しなかったことからホスホリパーゼA₂および血小板活性化因子加水分解酵素はPC-とPC-OHの分解にさほど重要ではないと考えられる。
血しょうの酸化における2種類の開始剤の違い, 高密度および低密度リポタンパク質の酸化の違いについても議論した。

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During the lipid peroxidation reaction, lipid hydroperoxides are formed as primary products. Several lines of evidence suggest that lipid hydroperoxides can trigger cell death in many cell types, including neurons. In a screening of lipid hydroperoxides which can induce toxicity in neuronal cells, we found docosahexaenoic acid hydroperoxides (DHA-) induced much severe levels of reactive oxygen species generation and cell death in human neuroblastoma SH-SY5Y cells compared to the hydroperoxides of linoleic acid and arachidonic acid. Therefore, we focused on DHA-, and demonstrated that DHA- apparently induced an apoptosis in the neuronal cells through several apoptotic hallmarks including nuclei condensation, DNA fragmentation, poly (ADP-ribose) polymerase cleavage and increased activity of caspase-3. We also found the signaling changes in mitochondria-mediated apoptosis, such as cytochrome c release and increased expression of Bcl-2, as well as a dose-dependent attenuation of mitochondrial membrane potential in the DHA- treated cells. These data indicated DHA hydroperoxide as a potential inducer of apoptosis in human neuroblastoma SH-SY5Y cells, which may be mediated by mitochondria dysfunction pathway.

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目的：ヒト皮膚は紫外線等の酸化ストレスに常に曝されており，皮脂のスクアレン（SQ）は太陽光暴露により酸化修飾を受けスクアレ ンモノハイドロペルオキシド（SQOOH）を生じることが報告されている。我々は太陽光暴露により6種類のSQOOH異性体が生成する ことを既に報告したが，各異性体がヒト皮膚細胞に及ぼす影響は明らかでない。そこで3次元培養ヒト皮膚モデルを用いて各異性体単 独あるいはそれらの混合物の細胞毒性及びサイトカイン産生能を検討した。
実験：SQOOHの6種類の異性体（2--SQ, 3--SQ, 6--SQ, 7--SQ,10--SQ, 11--SQ）及びそれらの混合 物（m-SQOOH）をVitro-life Skinに24時間暴露し，細胞毒性をテトラカラーワンで評価し，培地中のIL-1αをELISAで定量した。
結果・考察：m-SQOOHは20 mＭまでは細胞毒性は認められなかった。2--SQは5 mMで細胞生存率が有意に減少した。 2--SQはm-SQOOHよりも低濃度でIL-1αの産生を有意に増加させた。SQOOHは酸化修飾部位の違いにより細胞毒性及びIL-1 αの産生能が異なり，各SQOOH異性体のヒト皮膚中の存在量や他のサイトカイン産生能への影響等，今後更なる検討が必要と思われ た。

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Electron spin resonance (ESR) is regarded as the least ambiguous method for the detection of free radicals. Using spin trapping technique with 5, 5-dimethyl-pyrroline-N-oxide (DMPO) we measured superoxide generated by stimulated polymorphonulcear leukocytes (PMN) . The results were compared to those by chemiluminescence study with Cypridina luciferin analog (CLA) which is highly sensetive to superoxide. ESR spectrum was obtained by JEOL-JES-FE2XG ESR spectrometer. The intensity of the signal of DMPO- adduct was measured as ratio to the intensity of Mn²⁺ signal. Phorbol myristate acetate or opsonized zymosan were used as stimulants of PMN. The ESR signal of DMPO- was completely inhibited by superoxide dismutase, but not affected by catalase or sodium azide. The relative intensity of DMPO- signal and the maximal increase of CLA-dependent chemiluminescence were increased in proportion to the xanthine oxidase unit in hypoxanthine +xanthine oxidase system, and to the cell concentration in PMN system. There were positive correlation between the relative intensity of the ESR signal of DMPO- and CLA-dependent chemiluminescence by stimulatd PMN from patients. By ESR assay, increased generation of superoxide by PMN in patients with fairly controlled diabetes was shown.

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In order to elucidate the mechanism for the protective ability of final stable rust layer formed on a weathering steel by atmospheric corrosion, the ion selective property of rust layer was studied by the use of synthetic iron rust membranes. Anion selective property was increased in this order: Fe₃O₄<α-Fe₂O₃<α-FeOOH<γ-FeOOH<β-FeOOH. Fe₃O₄ has little effect on the ion selective property. Anion selective property decreased with increasing Cr content in α-(Fe_1-x, Cr_x) (Cr-substituted goethite), cation selective property came out when the Cr content exceeds 3.8 mass%. As the effect of adsorbed oxyanions, all of PO₄^3-, MoO₄^2- and SO₄^2- reduced the anion selective property or changed to cation selective property. Among these anions, PO₄^3- has significant effect on the ion selective property of rust membrane and suppresses the permeation of Cl^- through rust layer. In acidic solution, the ion selective property of α-(Fe_1-x, Cr_x) changed from cation to anion selective one, though little effect for α-FeOOH. The anion selective property of α-(Fe_1-x, Cr_x) was reduced as the concentration of KCl solution increased under the constant ratio of KCl solutions (C_I/C_II). Furthermore, it was thought that protective rust layer formed on a weathering steel acts as a kind of bipolar membrane which was composed of outer γ-FeOOH layer of anion selective property and inner α-(Fe_1-x, Cr_x) layer of cation selective property.

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