| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1999: ¥2,900,000 (Direct Cost: ¥2,900,000)
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| Research Abstract |
A simple and sensitive method using a high-performance liquid chromatography (HPLC) system with post-column reduction using platinum or zinc as the catalyst followed by electrochemical detection (ECD) has been developed for the quantification of the oxidation products of vitamin E (α-tocopherol, α-TH), including 8a-(phosphatidylcholine-dioxy)-α-tocopherones (TOO-PCs), 8a-(cholestrylester-dioxy)-α-tocopherones (TOO-ChEs), α-tocopherylquinone (TQ), and epoxy-α-tocopherylquinones (epoxyTQs). The lipids extracted from biological samples were treated with solid-phase extraction of a silica-NH_2 cartridge before the HPLC analysis ; THs, TQ, epoxyTQs, and TOO-ChEs were eluted with chloroform, TOO-PCs being with methanol, respectively. THs, TQ, and epoxyTQs in the chloroform fraction were separated by a J'sphere ODS-H80 column with 95% methanol containing 10 mM lithium acetate and 5 mM trifluoroacetic acid and quantified by ECD after on-line zinc-catalyzed reduction. TOO-PCs in the methanol fr
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action were separated by a J'sphere ODS-L80 column using 97.5% methanol containing 20 mM choline chloride, passed through a reduction column (5% platinum on alumina), and the reduced forms were quantified by ECD (+700 mV) ; TOO-ChEs in the chloroform fraction being the same conditions with 98.5% methanol containing 10 mM lithium acetate as the solvent. The detection limit for each compound by this method was about 0.2 pmol. This method was applied to the detection of the reaction products of a-TH in the peroxidized human plasma. The endogenous α-TH in plasma could scavenge peroxyl radicals of cholesterylester (ChE) and phosphatidylcholine (PC) to suppress the formation of ChE and PC hydroperoxides when the peroxidation was initiated by a free radical initiator at 37℃. The main products detected by reversed-phase HPLC were the addition products of TH with ChE-peroxyl radicals (TOO-ChE), TQ, and epoxyTQ.The structures of TOO-ChE were characterized as cholestryl (8a-dioxy-α-tocopherone)-epoxyoctadecenoates. The results indicate that endogenous α-TH in human plasma can react with lipid peroxyl radicals to form some reaction products.
In conclusion, the HPLC technique presented here is found to advantages for the sensitive and specific determination of the free-radical scavenging products of vitamin E in biological samples. Less
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