| Project/Area Number |
62570969
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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 | Kyushu University |
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Principal Investigator |
OHKURA Yosuke Faculty of Pharmaceutical Sciences, Kyushu University, Professor, 薬学部, 教授 (00037574)
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| Project Period (FY) |
1987 – 1988
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| Project Status |
Completed (Fiscal Year 1988)
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| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1988: ¥200,000 (Direct Cost: ¥200,000)
Fiscal Year 1987: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | Opioid peptide / Enkephalin / N-Terminal tyrosine-containing peptide / Brain / High-performance liquid chromatography / Selective fluorescence derivatization / Fluorescence detection / 高感度分析 / エンケフアリン / N末端ナロシン含有ペプチド / プレカラム蛍光誘導体化反応 / 高感度分析法の開発 |
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| Research Abstract |
High-performance liquid chromatographic method with fluorescence detection has been developed for the sensitive and selective determination of opioid peptides in rat brain. The method is beased on precolumn fluorescence derivatization specific for opioid peptides which have a tyrosyl residue at the N-terminus in their molecule.
The opioid peptides were extracted from the brain tissue by homogenization and deproteinization, and then passed through a reversed-phase mini-cartridge for cleanup. The extracted peptides were converted into fluorescent derivatives by reaction with hydroxylamine, cobalt(II) ion and borate in a weakly alkaline solution(pH 8.5).
The fluorescent derivatives of the peptides were separated on a reversed-phase column packed with TSKgel ODS-120T (support, silicagel) by gradient elution of acetonitrile in the mobile phase containing borate buffer(pH 8.5) and tetra-nbutylammonium chloride. The HPLC method with fluorescence detection could determine the endogenous opioid o
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ligopeptides such as leucine enkephalin (LEK) and methionine enkephalin (MEK) at concentrations as low as ca. 6 pmol per g of the brain tissues. However, some relatively large molecular opioid peptides such as neoendorphins and dynorphins were not be eluted from the column.
In the further course of the investigation, the fluorescent derivatives of the large opioid peptides could be separated on an Asahipak ODP-50 column (support, synthetic hard polymer) by the HPLC. The endogenous opioid peptides of LEK, MEK, MEK-ARG-PHE and MEK-ARG-GLY-Leu were simultaneously quantified by the modified HPLC method. The lower limits (S/N=2) of detection for these peptides in the tissue were 8.4-47 pmol per g of the tissue, which corresponded to 0.27-2.8 pmol per a 100-ul injection volume. The developed HPLC method could be readily applied to the identification of the peptide in the tissue by means of the enzymatic degradation of the sample.
This research project provided the first practical HPLC method with fluorescence detection for the determination of the endogenous opioid peptides in rat brain. Less
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