Measurement of Lipid Hydroperoxides by the Ferric-Xylenol Orange Method (2) : Application to Lipoxygenase Assay

スポンサーリンク

概要

• 論文の詳細を見る

• The ferric-xylenol orange (FOX) method has been used for quantification of hydroperoxides by measuring the colored ferric-xylenol orange (XO/Fe^<3+>) product with a peak absorbance at 560nm. We recently reported a modified FOX method, the sensitivity of which was increased in the presence of membranous phosphatidylcholine (PC) by forming a XO/Fe^3+-PC complex with a peak absorbance at 610nm. Lipoxygenases (LOXs) and their metabolites have been implicated in a wide range of disease states. We applied our newly developed FOX method to the assay of human 15-lipoxygenase-2 (15-LOX-2) and soybean lipoxygenase (SLOX) as typical animal and plant lipoxygenases, respectively. The amounts of 15-S-hydroperoxyeicosa-5,8,10,14-tetraenoic acid (15-HPETE) produced by 15-LOX-2 measured by UV absorption at 237nm attributed to the conjugated diene, coincided with the results of our FOX method measuring absorbance at 610nm. The 15-HPETE production time courses measured by the two methods also correlated well. SLOX rapidly oxidized unesterified linoleic acids (LA) and slowly esterified fatty acids in egg yolk PC (EYPC). Availability of EYPC was increased if the membrane structure was moderately disturbed by MeOH and Triton X-100, but LA oxidation was readily decreased by them. These results indicate that our method is useful for lipoxygenase assay. Furthermore, our method was applicable to assaying the inhibitory effect of 5,8,11,14-eicosatetraynoic acid (ETYA) on SLOX activity using LA and EYPC as substrates. The inhibition dose-dependency of ETYA was almost the same in the LA and EYPC systems, although the enzyme concentrations differed by a factor of 1,000, suggesting that ETYA functioned as a competitive inhibitor. These results indicate that our method may be useful as a screen for the identification of novel inhibitors of lipoxygenases.

著者

• FUKUZAWA Kenji

  Faculty of Pharmacy, Yasuda Women's University

• SANO Masumi

  Graduate School of Pharmaceutical Sciences, The University of Tokushima

• AKAI Kaori

  Graduate School of Pharmaceutical Sciences, The University of Tokushima

• MORISHIGE Junichi

  Graduate School of Pharmaceutical Sciences, The University of Tokushima

• TOKUMURA Akira

  Graduate School of Pharmaceutical Sciences, The University of Tokushima

• JISAKA Mitsuo

  Faculty of Life and Environmental Science, Shimane University

• Akai Kaori

  Graduate School Of Pharmaceutical Sciences The University Of Tokushima

• Sano Masumi

  Graduate School Of Pharmaceutical Sciences The University Of Tokushima

• Jisaka Mitsuo

  Faculty Of Life And Environmental Science Shimane University

• Fukuzawa Kenji

  Division Of Physiological Chemistry Shibaura Institute Of Technology

• Fukuzawa K

  Fac. Of Pharmacy Yasuda Women's Univ.

• Fukuzawa Kenji

  Faculty Of Pharmacy Yasuda Women's University

• Morishige Junichi

  Graduate School Of Pharmaceutical Sciences The University Of Tokushima

• Fukuzawa Kenji

  Faculty Of Pharmaceutical Sciences The University Of Tokushima

• Tokumura Akira

  Graduate School Of Pharmaceutical Sciences The University Of Tokushima

• Jisaka Mituo

  Faculty of Life and Environmental Science, Shimane University

関連論文

• Inhibitory effect of Lysichiton camtschatcense extracts on Fe^/ascorbate-induced lipid peroxidation in rat kidney and brain homogenates
• Measurement of Lipid Hydroperoxides by the Ferric-Xylenol Orange Method (2) : Application to Lipoxygenase Assay
• Depolarization-induced Rapid Generation of 2-Arachidonoylglycerol, an Endogenous Cannabinoid Receptor Ligand, in Rat Brain Synaptosomes
• Influence of Oxidative Stress on Fusion of Pre-Synaptic Plasma Membranes of the Rat Brain with Phosphatidyl Choline Liposomes, and Protective Effect of Vitamin E
• Measurement of Lipid Hydroperoxides by the Ferric-Xylenol Orange Method (1) : Characteristics of the Ferric-Xylenol Orange/Membrane Phosphatidylcholine Complex
• Dynamics of Lipid Peroxidation and Antioxidion of α-Tocopherol in Membranes
• α-Tocopheryl Succinate Activates Protein Kinase C in Cellular and Cell-Free Systems
• Cytotoxicity of α-Tocopheryl Succinate, Malonate and Oxalate in Normal and Cancer Cells In Vitro and Their Anti-Cancer Effects on Mouse Melanoma In Vivo
• Vitamin E Depletion Enhances Liver Oxidative Damage in Rats with Water-Immersion Restraint Stress

スポンサーリンク