2002 Fiscal Year Final Research Report Summary
Modified structure and biological activities for oxidized LDL present in vivo searching for a better model for the physiological oxidized LDL
| Project/Area Number |
13672303
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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 | Teikyo University |
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Principal Investigator |
ITABE Hiroyuki Teikyo Univ. , Fac. of Pharmaceutical Sciences,Associate Professor, 薬学部, 助教授 (30203079)
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| Project Period (FY) |
2001 – 2002
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| Keywords | oxidized LDL / oxidized phosphatidylcholine / minimally modified LDL / atherosclerosis / HPLC / monoclonal antibody / macrophages |
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| Research Abstract |
It is widely believed that oxidized LDL (OxLDL) is involved in atherogenesis. We have demonstrated the presence of OxLDL in circulating plasma by developing a sensitive method of measuring OxLDL using a monoclonal antibody. Recently, a question arises whether copper-induced oxidation of LDL, which has been widely utilized as a model, reflects the characteristics of OxLDL in vivo. An alternative model for in vivo OxLDL is called minimally modified LDL (MM-LDL) which is prepared under milder conditions. In the present study, various modified LDLs were characterized.
MM-LDL was prepared by the method described by Berliner et al. , in which LDL was dialyzed against PBS containing FeS04 at 4 ℃ for 4 days. While the MM-LDL contained a quarter as much of TBARS compared to copper-induced OxLDL, the amount of conjugated dienes and reactivity to anti-oxidized phosphatidylcholine (OxPC) antibody in the both modified LDLs were almost the same. When aldehyde-containing OxPC in the total lipids extracted from the modified LDLs, 10-fold larger amount of aldehyde-containing OxPC was present in MM-LDL than copper-induced OxLDL. From these results, it is clear that oxidized products and modified structures in the oxidatively modified LDL can be varied depending on the treatment.
The next step for this study is to compare the oxLDL present in vivo with these modified LDL models. In order to do this, a procedure to isolate minute amount of OxLDL from plasma and sensitive methods to determine oxidation products must be improved. As low as 10 pmol of aldehyde-containing OxPC can be detected as fluorescent derivatives, however, even more sensitive analysis was achieved using ESI/MS procedure. Isolation of OxLDL from plasma was not successful so far by ion-exchange HPLC or density gradient ultracentrifugation. We are currently trying an immuno-precipitaion procedure for this purpose.
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[Publications] Higashi, Y., Itabe, H., Fukase, H., Mori, M.,Fujimoto, Y., Sato, R., Imanaka, T. and Takano, T: "Distribution of microsomal triglyceride transfer protein within sub-endoplasmic reticulum regions in human hepatoma cells"Biochim. Biophys. Acta. 1581. 127-136 (2002)
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[Publications] Ehara, S., Ueda, M., Naruko, T., Haze, K., Ogami,M., Ikura, Y., Itabe, H., Komatsu, R., Yoshiyama, M.,Takeuchi, K. and Yoshikawa, J.: "Pathophysiological role of oxidized low-density lipoprotein in plaque instability in coronary artery diseases"J. Diabetes Compl.. 16. 60-64 (2002)
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[Publications] Nishi, K., Itabe, H., Uno, M., Kitazato K. T.,Horiguchi, H., Shinno, K. and Nagahiro S.: "Oxidized LDL in carotid plaques and plasma associates with plaque instability"Arterioscler. Thromb. Vasc. Biol.. 22. 1649-1654 (2002)
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[Publications] Azumi, H., Inoue, N., Ohashi, Y., Terashima, M.,Mori, T., Fujita, H., Awano, K., Kobayashi, K., Maeda,K., Hata, K., Shinke, T., Kobayashi, S., Hirata, K.,Kawashima, S., Itabe, H., Hayashi, Y., Imajo-Ohmo, S.,Itoh, H. and Yokoyama, M.: "Superoxide generation in directional coronary atherectomy specimens of patients with angina pectris. Important role of p22phox-based NADH/NADPH oxidase"Arterioscler. Thromb. Vasc. Biol.. 22. 1838-1844 (2002)
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