| Project/Area Number |
16590065
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biological pharmacy
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| Research Institution | Showa University |
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Principal Investigator |
ITABE Hiroyuki Showa University, School of Pharmaceutical Sciences, Professor, 薬学部, 教授 (30203079)
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| Co-Investigator(Kenkyū-buntansha) |
KATO Rina Showa University, School of Pharmaceutical Sciences, Research, 薬学部, 助手 (30392400)
OBAMA Takashi Showa University, School of Pharmaceutical Sciences, Research, 薬学部, 助手 (60395647)
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2005: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2004: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | oxidized LDL / atherosclerosis / monoclonal antibody / apoE-knockout mouse / mass spectrometry / oxidative stress / LDL |
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| Research Abstract |
We have proved the occurrence of oxidized low-density lipoprotein (OxLDL) in human circulating plasma through establishing a sensitive method to measure OxLDL using a monoclonal atnibdy (DLH3) recognizing oxidized phosphatidylcholine. OxLDL, known for a risk factor for atherosclerosis, is a mixture of heterogeneously modified particles. The precise nature of OxLDL present in vivo has not yet been elucidated. In this study we tried to separate OxLDL from human plasma and analyze its modified structures. Elucidation of in vivo OxDL itself should be a crucial step for understanding metabolic behavior and clinical significance of OxLDL in vivo.
It was estimated previously, based on the sandwich ELISA using DLH3 antibody, that there is approximately one OxLDL particle in 10000 LDL particles in human plasma. To analyze such minute amount of materials, we introduced ESI-LC/MS/MS technique. Human LDL pre-treated either with acrolein or 4-hydroxynonenal (HNE) was trypsin-digensted, then the samole was subjected to LC/MS/MS analysis. We successfully detected and identified tryptic fragments of apoB containing acrolein-modified lysine or HNE-modified histidine. These typical modified structures are good markers for oxidative modification of proteins, and they are useful for analysis of OxLDL in vivo. We also successfully detected oxidized phospholipids and oxidized cholesterols using MS/MS. It is very likely that effective concentration step of OxLDL from plasma is needed to analyze plasma OxLDL. Separation of OxLDL in plasma using an OxLDL-absorbing dialysis membrane in a preliminary trial, that and identified tryptic fragments of apoB containing acrolein-modified lysine or HNE-modified histidine. modified with
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