1989 Fiscal Year Final Research Report Summary
Development and Application of High-performance Liquid Chromatography/Chemiluminescence Determination of Biological Thiols
| Project/Area Number |
63571023
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Physical pharmacy
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| Research Institution | Nagasaki University |
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Principal Investigator |
NAKASHIMA Kenichiro Nagasaki University, School of Pharmaceutical Sciences, Associate Professor, 薬学部, 助教授 (30039656)
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| Co-Investigator(Kenkyū-buntansha) |
AKIYAMA Shuzo Nagasaki University, School of Pharmaceutical Sciences, Professor, 薬学部, 教授 (50028148)
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| Project Period (FY) |
1988 – 1989
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| Keywords | Biological thiols / High-performance liquid chromatography / Chemiluminescence detection / Aryl oxalate / Lipoperoxide / Malondialdehyde / Peroxyoxalate chemiluminescence |
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| Research Abstract |
The results obtained in this study are follows:
1. A high-performance liquid chromatography (HPLC)-chemiluminescence (CL) determination method of biological thiols using N-[4-(6-dimethylamino-2-benzofuranyl)phenyl]maleimide (DBPM) as a fluorogenic label has been developed and applied to the assay of reduced glutathione and cysteine in rat liver. The method was very sensitive and could detect fmol levels of thiols. Next, another HPLC-CL method using 4-fluoro-7-nitrobenzo-2,1,3-oxadiazole (NBD-F) as a label was developed. However, the sensitivity of the method was lower and pmol level. In the course of this study, it was found that CL detection was not always sensitive than fluorescence detection for some thiols. It might be caused by the suitabilities of the chemiluminogenic reagents used.
2. To evaluate oxalates and fluorescers as peroxyoxalate CL reagents, a simple and rapid method by flow system has been developed. By the method, newly synthesized aryl oxalates and fluorescent compounds were evaluated and found some useful compounds as CL reagents. The method should be suitable for the selection of HPLC-peroxyoxalate CL reagents.
3. By the method of 2, CL efficiencies of condensates of malondialdehyde (MDA) with thiobarbituric acids were determined. As the result, MDA-1,3-diethyl-2-thiobarbituric acid (DETBA) condensate showed the largest CL efficiency and could be detect as low as 10 fmol. This suggests that DETBA would be an excellent derivatization reagent for MDA. By using DETBA, a high sensitive assay method for lipoperoxide (MDA) should be developed.
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