Different Rates of Geometric Isomers of Linoleate Hydroperoxide in Acid-catalyzed Decomposition

Methyl linoleate hydroperoxide was decomposed with 0.1 m HCl in acetone-water (9:1, v/v) at 30°C. The decrease in four isomers of the hydroperoxide was monitored by HPLC without any derivatization. In both isomers having 13- and 9-hydroperoxy groups, those having trans, trans dienes decomposed more rapidly than those having cis, trans dienes. In all the isomers, the rates of decomposition were first order with respect to concentrations of the hydroperoxides. The yields of 2-nonenal and 12-oxo-10-dodecenoate were also measured by GC-MS. 12-Oxo-10-dodecenoate was produced only from the 13-isomers and 2-nonenal from the 9-isomers. The rapid decomposition of the trans, trans isomers didn’t seem to be responsible for the formation of these aldehyde products.     

Methyl Linoleate Dimer and Methyl 8-Phenyloctanoate in the Copper-catalyzed Decomposition Products of Methyl Linoleate Hydroperoxides

Cochliophilin A (5-hydroxy-6,7-methylenedioxyflavone, 1), known as a host-specific attractant towards the zoospores of Aphanomyces cochlioides, a cause of root rot and damping-off diseases of Chenopodiaceae, was found in the Amaranthaceae plant, Celosia cristata, that is susceptible to the pathogen. The content of 1 in Celosia seedlings was quantified as 1.4 μg/g fresh weight. A new isoflavone, cristatein (5-hydroxy-6-hydroxymethyl-7,2′-dimethoxyisoflavone, 2), and five known flavonoids were also identified.     

Influence of Linoleate Hydroperoxide Minor Isomers on GLC Analysis of Oleate Hydroperoxides

The distribution of the methylcitric acid cycle and the modified ^-oxidation pathway for propionate catabolism was surveyed in yeasts and filamentous fungi, mainly by comparing the activities of the key enzymes. All the six tested species of filamentous fungi belonging to five genera and 21 species of yeasts belonging to eleven genera were found to catabolize propionate through the methylcitric acid cycle, with the exception of Candida rugosa and one group of strains of C. catenulata, which catabolize propionate through the ß-oxidation pathway. From the observed diversity of propionate catabolism among closely related strains or species, it was assumed that different minor pathways evolved from universal metabolic pathways, such as the citric acid cycle and the ^-oxidation pathway for fatty acids, in later stages of an evolutionary history.     

非水介质中脂肪酶催化亚油酸油醇酯合成的研究

利用实验室自制及购买的几种脂肪酶制剂催化的酯化反应来制备亚油酸油醇酯,其中本实验室制备的丝孢酵母脂肪酸酯化效果最好,作为进一步研究的实验用酶。以正己烷为反应溶剂,在微水系统中对影响亚油酸油醉酯合成的各种因素进行了研究,确定酯化反应合成的最适温度为３５℃,０～１００℃反应１０ｈ的酯化率均可达到９０％,最适酯化ｐＨ为８．０,最适底物浓度为０．２５ｍｏｌ／Ｌ,最适水含量为０．０５％,在选用的１１种有机溶剂中,以环己烷的酯化率最高,二甲亚砜最低。     

The Photoxidative Alteration of Chlorophylls in Methyl Linoleate and Prooxidant Activity of Their Decomposition Products

Methyl linoleate containing chlorophylls and/or pheophytins was exposed to light in the presence of oxygen. The photooxidative reaction of both chlorophylls a and b was first-order, and the reaction rate for chlorophyll a was higher than that for chlorophyll b. On the other hand, pheophytins a and b hardly decomposed even after irradiation for 24 hr, and retained a green or a brownish-green color. In qualitative analysis of the photooxidation products of chlorophylls a and b, no pheophytins or pheophorbides were detected, while green and polar red pigments were observed on a thin layer chromatogram near the spot of chlorophyll and the origin, respectively. These photooxidation compounds also had prooxidant effects as well as did chlorophyll.     

Generation of Free Radicals during Decomposition of Hydroperoxide in the Presence of Myeloperoxidase or Activated Neutrophils

It was shown with the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone that myeloperoxidase (MPO) in the presence of its substrates H2O2 and Cl- as well as activated neutrophils destroy tert-butyl hydroperoxide producing two adducts of O-centered radicals which were identified as peroxyl and alcoxyl radicals. Inhibitory analysis performed with traps of hypochlorite (taurine and methionine), free radical scavengers (2,6-di-tret-butyl-4-methylphenol and mannitol), and MPO inhibitors (salicylhydroxamic acid and 4-aminobenzoic acid hydrazide) revealed that the destruction of the hydroperoxide group in the presence of isolated MPO or activated neutrophils was directly caused by the activity of MPO: some radical intermediates appeared as a result of the chlorination cycle of MPO at the stage of hypochlorite generation, whereas the other radicals were produced independently of hypochlorite, presumably with involvement of the peroxidase cycle of MPO. The data suggest that the activated neutrophils located in the inflammatory foci and secreting MPO into the extracellular space can convert hydroperoxides into free radicals initiating lipid peroxidation and other free radical reactions and, thus, promoting destruction of protein-lipid complexes (biological membranes, blood lipoproteins, etc.).     

Kinetics of Inhibition of Horse Liver Alcohol Dehydrogenase byp-Methylbenzyl Hydroperoxide

Abstract

The complex kinetic behaviour of p-methylbenzyl hydroperoxide in its inhibitory action on horse liver alcohol dehydrogenase was examined. The kinetic patterns are markedly different at very low (<10^?8 M) and high (> 10^?7 M) hydroperoxide concentrations. In both cases very low inhibition constants (4nM and 14nM, respectively) were found. A possihle mechanistic model based on these results is discussed.     

Geometrical Isomers of Monohydroperoxides Formed by Autoxidation of Methyl Linoleate

Isomeric monohydroperoxides produced from autoxidized methyl linoleate were separated into two geometrical isomers (cis-trans and trans-trans) by silver nitrate TLC. Purified monohydroperoxides were converted into hydroxy octadecadienoates. Trimethylsilyl (TMS) derivatives of these compounds (four components) were separated into three peaks in the gas chromatogram; the mixture of 9-hydroxy-cis,trans-isomer and 13-hydroxy-cis,trans-isomer, 9-hydroxy-trans,trans-isomer and 13-hydroxy-trans,trans-isomer. The trans-trans isomers became more dominant than the cis-trans isomers in the later stage of autoxidation and with the rise of temperature. At the degradation of monohydroperoxides, the decrease of trans- trans isomers was apparently slower than that of cis-trans isomers. It is proposed that cis,trans isomerization of monohydroperoxides takes place at the process of autoxidation of methyl linoleate.     

Detection of DNA Damage in Cultured Human Fibroblasts Induced by Methyl Linoleate Hydroperoxide

To establish a strategy to generate N-acylated proteins modified with fatty acids having a specific chain length, tGelsolin-streptag, an epitope-tagged model protein having an N-myristoylation motif, was synthesized using an insect cell-free protein synthesis system in the presence of acyl-CoA with various fatty acid chain lengths. It was found that the fatty acid species attached to the N-termini fully depended on the acyl-CoA species added to the reaction mixture. N-Acylated proteins with fatty acid chain lengths of 8, 10, 12, and 14 were generated successfully.     

磷脂酰胆碱过氧化物的测量和生化机理及其在动脉粥样硬化中的应用(英文)

磷脂酰胆碱过氧化物(phosphatidylcholine hydroperoxide,PCOOH)是磷脂酰胆碱(phosphatidylcholine,PC)氧化的最初产物,在包括动脉粥样硬化在内的各种病理条件下,可以在血浆和组织中检测到。为了评定动脉粥样硬化的程度,我们研究了PCOOH对THP-1细胞与内皮细胞黏附分子(intracellular adhesionmolecule-1,ICAM-1)之间粘附状态的影响,发现THP-1细胞与内皮细胞黏附分子的粘附是剂量依赖于PCOOH的。不氧化的PC、sn-2截断的PC和其他过氧化物不影响THP-1细胞与内皮细胞黏附分子的黏附。在PCOOH处理的细胞中,发现了F-肌动蛋白富集的突出膜结构,与淋巴细胞功能关联的抗原(lymphocytefunction-associated antigen-1,LFA-1)定位在突出结构上。细胞松弛素D和肌动蛋白聚合抑制剂能够抑制PCOOH诱导细胞黏附到ICAM-1和膜突起上。我们研究了参与PCOOH诱导THP-1细胞黏附到ICAM-1上的Rho-家族的GTP酶,发现氟伐他汀对异戊二烯的消耗以及GGTI-286对牛儿基转移酶的阻害均能够抑制PCOOH诱导细胞黏附到ICAM-1和膜上。Pull-down方法表明,在PCOOH处理的细胞中,Rac1和Rac2被活化。Pan-Rho-家族的GTP酶抑制剂难辨梭状芽孢杆菌B、Rac特异抑制剂NSC23776和Rac同型体的RNA干扰,均能够减少细胞黏附。这些结果表明,PCOOH诱导的LFA-1调节的细胞黏附到ICAM-1上是通过actin细胞骨架。这一机理可能参与了单核细胞黏附到动脉壁上并启动了动脉粥样硬化。     

茶树脂氢过氧化物裂解酶基因CsiHPL1的克隆及表达

根据茶树基因CsiHPL1的cDNA序列设计引物,采用RT-PCR方法从茶树品种龙井43′中克隆了CsiHPL1序列,并分析了CsiHPL1在生物和非生物胁迫下的诱导表达情况及亚细胞定位。结果表明,CsiHPL1包含一个1 476 bp的最大开放阅读框,编码491个氨基酸,预测为13-HPL基因。Real-Time PCR分析结果表明,CsiHPL1的表达受到茶尺蠖取食、机械损伤和茉莉酸的诱导;构建了CsiHPL1与绿色荧光蛋白（GFP）基因的重组表达载体,经农杆菌瞬时转化烟草叶片,利用激光共聚焦显微镜在叶绿体中观察到GFP荧光,表明CsiHPL1编码的蛋白质为叶绿体蛋白质。     

Cleavage of α-Dicarbonyl Compounds by Terpene Hydroperoxide

Thermostabilities of component enzymes in the pyruvate dehydrogenase complex from Bacillus stearothermophilus decreased in the order lipoamide dehydrogenase, lipoate acetyltransferase, and pyruvate decarboxylase (E1). Fluorescence of an extrinsic 8-amino-1-naphthalenesulfonate (ANS) increased with inactivation of E1. The thermal denaturation of the enzymes resulted in disassembly of the complex. El was involved in a resulting aggregate of the complex. The interaction between ANS and denatured E1 accounted for an increase in fluorescence.     

Linoleate isomerase activity occurs in lactic acid bacteria strains and is affected by pH and temperature

Aims: To investigate the ability of lactic acid bacteria (LAB) to convert linoleic acid (LA) and α‐linolenic acid (α‐LNA) to conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA), respectively. To assess pH and temperature influences on CLA and CLNA production by Lactobacillus sakei LMG 13558. Methods and Results: A screening of 48 LAB yielded one Lactobacillus curvatus, five Lactobacillus plantarum and four Lact. sakei strains displaying linoleate isomerase (LAI) activity. CLNA conversion percentages varied largely (1–60%). CLA conversion, occurring in three strains, was lower (2–5%). The LAI gene sequences of the ten LAI‐positive strains shared 75–99% identity with the LAI gene sequence of a Lact. plantarum AS1.555. At pH 6·2, CLA and CLNA production by Lact. sakei LMG 13558 was higher at 30°C than at 20 and 25°C. At pH 5·5 (30°C) or 37°C (pH 6·2), LA was not converted and α‐LNA only slightly converted. Conclusions: LAB show strain‐dependent LAI activity. Production of CLA and CLNA is affected by pH and temperature, as shown for Lact. sakei LMG 13558. Significance and Impact of the Study: Several LAB produce CLA and/or CLNA, as shown for Lact. sakei and Lact. curvatus for the first time. These findings offer potential for the manufacturing of fermented functional foods.     

Partial purification and characterization of the linoleate hydroperoxide isomerase from grains of Hordeum distichum

Linoleate hydroperoxide isomerase was mainly located in the embryos of barley grains and its activity decreased during germination. The enzyme partially purified from embryos converted 9-hydroperoxy, trans-10, cis-12-octadecadienoic acid to 9-hydroxy, 10-oxo, cis-12-octadecenoic acid and 13-hydroxy, 10-oxo, trans-11-octadecenoic acid in the ratio of ca 2:1.     

Protective Effect of Phenolic Antioxidants on the Cytotoxicity Induced by Phosphatidylcholine Hydroperoxide

Phosphatidylcholine hydroperoxides show weak but distinct toxicity toward cultured human umbilical vein endothelial cells. The protective effect of phenolic antioxidants against the cytotoxicity of phosphatidylcholine hydroperoxides was examined. Probucol depressed the toxicity most effectively among the antioxidants studied under both pretreatment and concurrent treatment conditions. α-Tocopherol showed a protective effect in the case of concurrent treatment. Protection by phenolic antioxidants against the cytotoxicity of phosphatidylcholine hydroperoxides seems to depend on their incorporation rate into cells, their affinity for phospholipids, their antioxidative activity, and their orientation in membranes.     

Potentiation and Inhibition of Ionotropic Neurotransmitter Receptors Expressed in Xenopus Oocyte by Linoleic Acid and Its Hydroperoxide

Abstract: To study the effects of lipid hydroperoxide on ionotropic neurotransmitter receptors, γ-aminobutyric acid (GABA), N -methyl- d -aspartate (NMDA), and non-NMDA receptors (GABARs, NMDARs, and non-NMDARs, respectively) were expressed in Xenopus oocytes that received an injection of mRNA prepared from rat whole brain. Linoleic acid (LA) and its hydroperoxide 13- l -hydroperoxylinoleic acid (LOOH) prepared with soybean lipoxygenase inhibited the response of GABARs in the presence of GABA at high concentrations. The inhibition was stronger when the inhibitors were perfused 1 min before a mixture of GABA and the inhibitors than when they were perfused simultaneously with GABA. On the other hand, only LOOH potentiated the response of GABARs in the presence of GABA at low concentrations, possibly increasing the affinity of GABA to the receptors. Both LA and LOOH accelerated the rate of desensitization of GABARs, but LOOH did not affect their equilibrium between the active and desensitized form of the receptors. They also inhibited the response of NMDARs in a noncompetitive manner but barely inhibited the response of non-NMDARs in the presence of kainate at various concentrations. These results suggest the possibility that production of lipid hydroperoxide modulates the neural transmission in the brain, especially through GABARs.     

高效液相色谱测定微量胆固醇氧化产物

介绍一种高效液相色谱测定胆固醇氧化产物的方法,以苯甲酰氯为衍生剂,将胆固醇及其氧化产物衍生成苯甲酸酯,反相高效液相色谱分离,紫外检测,内标法定量．本法灵敏度高,重现性好,可应用于胆固醇纯度标准物质的杂质分析及血清中的胆固醇氧化产物测定。     

苦瓜谷胱甘肽磷脂氢过氧化物酶cDNA的克隆及其特征分析

根据谷胱甘肽磷脂氢过氧化物酶(PHGPX)氨基酸序列中高度保守的区段设计引物,采用RACE-PCR从苦瓜中克隆到一个全长927 bp的cDNA片段.DNA序列的数据库分析比较表明,该cDNA编码167个氨基酸,含有动植物PHGPX的特征结构,是一个新发现的苦瓜PHGPX基因（mocPHGPX）.RNA印迹结果显示,该基因在苦瓜幼苗的根中表达相对较弱,茎的信号较强,叶中最强.这些结果将有助于深入研究植物PHGPX的功能以及全面了解植物抗氧化体系.     

Pro/antioxidant Status in Murine Skin Following Topical Exposure to Cumene Hydroperoxide Throughout the Ontogeny of Skin Cancer

Organic peroxides used in the chemical and pharmaceutical industries have a reputation for being potent skin tumor promoters and inducers of epidermal hyperplasia. Their ability to trigger free radical generation is critical for their carcinogenic properties. Short-term in vivo exposure of mouse skin to cumene hydroperoxide (Cum-OOH) causes severe oxidative stress and formation of spin-trapped radical adducts. The present study was designed to determine the effectiveness of Cum-OOH compared to 12-O-tetradecanoylphorbol-13-acetate (TPA) in the induction of tumor promotion in the mouse skin, to identify the involvement of cyclooxygenase-2 (COX-2) in oxidative metabolism of Cum-OOH in keratinocytes, and to evaluate morphological changes and outcomes of oxidative stress in skin of SENCAR mice throughout a two-stage carcinogenesis protocol. Dimethyl-benz[a]anthracene (DMBA)-initiated mice were treated with Cum-OOH (32.8 micro mol) or TPA (8.5 nmol) twice weekly for 20 weeks to promote papilloma formation. Skin carcinoma formed only in DMBA/Cum-OOH-exposed mice. Higher levels of oxidative stress and inflammation (as indicated by the accumulation of peroxidative products, antioxidant depletion, and edema formation) were evident in the DMBA/Cum-OOH group compared to DMBA/TPA treated mice. Exposure of keratinocytes (HaCaT) to Cum-OOH for 18 h resulted in expression of COX-2 and increased levels of PGE(2). Inhibitors of COX-2 efficiently suppressed oxidative stress and enzyme expression in the cells treated with Cum-OOH. These results suggest that COX-2-dependent oxidative metabolism is at least partially involved in Cum-OOH-induced inflammatory responses and thus tumor promotion.     

Gas Chromatography Mass Spectrometric Analysis of Monohydro-peroxide Isomers and Their Secondary Oxidation Productsof Methyl Linoleate and Methyl Linolenate

Emulsions of methyl linoleate monohydroperoxides (18:2-monoHP) and methyl linolenate monohydroperoxides (18: 3-monoHP) were incubated with ferrous sulfate and ascorbic acid. Gas chromatography mass spectrometric analysis of the trimethylsilyl and te^butyldimethylsilyl derivatives of the reaction products showed that isomerization and secondary oxidation happen competitively during decomposition of 18:2-monoHP, while the secondary oxidation reaction proceeds preferentially and little isomerization is observed in 18: 3-monoHP. It is suggested that 18:3-monoHP is more susceptible to secondary oxidation than 18:2-monoHP because of 18:3 specific secondary oxidation resulting in hydroperoxy-cyclic peroxides and dihydroperoxides. Moreover, an experiment using ¹⁸0₂ has demonstrated that molecular oxygen is scrambled by isomerization and secondary oxidation. It was confirmed that molecular oxygen is attached preferentially to the C-13 position in the 9-monoHP isomer and C-9 position in the 13-monoHP isomer during degradation of 18:2-monoHP.