| Project/Area Number |
12556020
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
食品科学・栄養科学
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| Research Institution | The University of Tokushima |
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Principal Investigator |
TERAO Junji The University of Tokushima, School of Medicine, Professor, 医学部, 教授 (60093275)
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| Co-Investigator(Kenkyū-buntansha) |
MOON Ae-hak The University of Tokushima, School of Medicine, Associate Professor, 医学部, 助手 (20322258)
MUROTA Kaeko The University of Tokushima, School of Medicine, Associate Professor, 医学部, 助手 (40294681)
YAMANISHI Rintaro The University of Tokushima, School of Medicine, Associate Professor, 医学部, 助教授 (30253206)
TAKI Takao Otsuka, Institute for Molecular Science, Pharmacy. Co. Director, 分子科学研究所, 所長(研究職) (10046295)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2002: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2001: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | peroxidized lipids / TLC blot / atherosclerosis / denatured LDL / lipid hydroperoxides / plasma lipids / cholesteryl ester / phospholipids |
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| Research Abstract |
A blotting technique was developed to specifically detect lipid hydroperoxides in thin-layer chromatography. Phosphatidylcholine hydroperoxides (PC-OOH) and cholesteryl linoleate hydroperoxides (CE-OOH) ranging from 0.2 to 0.5 nmol, which were prepared by reaction with soybean lipoxygenase, were visualized as fluorescence spots on the blotted membranes by immersing the plate into a blotting solvent containing diphenyl-1-pyrenyphosphine (DPPP). This technique was applied successfully to monitor lipid peroxidation in human low-density lipoprotein (LDL) in vitro. We applied this methodology for convenient isolation of LOOH from biological samples and its derivatization to hydroxyl compound. As a first step, analysis of CE-OOH in human LDL was performed by the combination of TLC blotting and GC-MS, because oxidized LDL has been implied as a possible biomarker for the development of atherosclerosis. Human LDL was oxidized with copper ion (CuS04), a water-soluble azo radical generator (AAPH)
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, photosensitizer +light, or 15-lipoxygenase (15-LOX). After lipid extraction, samples were applied to TLC blotting with the solvent containing DPPP. The fluorescent spot corresponding to standard CE-OOH was removed and extracted as hydroxyl derivative of CE-OOH Then the samples were subjected to GC-MS/SIM analysis after additional derivatization involving hydrogenation, transmethylation and trimethylsilylation. By these procedures, isomeric CE-OOH accumulated in the LDL were determined as trimethylsilyl derivatives of methyl hydroxyoctadecanoate. In each case, we succeeded in the determination of isomeric CE-OOH produced from the oxidation of LDL. The methodology described here serves as a convenient and sensitive technique for the determination of isomeric LOOH from biological samples. In the case of application for antioxidant activity, analysis by the TLC blot technique showed that the gastric mucosa has the highest potential to eliminate PC-OOH. Furthermore the fact that free fatty acid hydroperoxides detected in blotted membranes strongly suggested the participation of phospholipase A2 and GSH-peroxidase in the detoxification of PC-OOH by gastric mucosa. Less
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