A chemiluminescent assay for hydroperoxide level of phosphatidylcholine hydroperoxide fraction purified by two Sep-Pak cartridges in biological samples

Kazuhiko Seya, Nobuhiro Ohkohchi, Hiroshi Shibuya, Masahide Satoh, Kosei Oikawa, Tatsuya Fukumori, Susumu Satomi, Shigeru Motomura

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5 Citations (Scopus)

Abstract

A chemiluminescent assay for hydroperoxide level of phosphatidylcholine hydroperoxide (PCOOH) fraction purified from biological samples was presented. This method utilized of two Sep-Pak cartridges. A lipid soluble fraction was isolated from each homogenized tissue or blood by Folch's method. The mixture of phosphatidylcholine (PC) and PCOOH was separated from the lipid soluble fraction by a Sep-Pak silica cartridge. A Sep-Pak tC18 cartridge made complete separation of both PCOOH and PC possible. The hydroperoxide level of PCOOH fraction was quantified by the reaction with ferrous ion using 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo[1,2-a]pyrazin-3-one as a chemiluminescent dye. The mixture of positional isomers, 1-hexadecanoyl-2-[9, or 10-hydroperoxyl octadecanoyl]-sn-glycero-3-phosphocholine was used as an authentic standard. The good recovery rate for authentic PCOOH of 87.1±11.6% (mean±S.E., n=4) was obtained by using two Sep-Pak cartridges. Linear calibration curve was obtained in the range from 2.5 to 20 nmol, and the detection limit of the standard was 10 pmol (signal-to-noise ratio>3). This method was applied to the investigation of the lipid peroxidation induced by reperfusion of the liver with cold preservation, mimicking liver transplantation in rats. The effect of liposome-encapsulated dichloromethylene diphosphonate (LEDD), which eliminate of Kupffer cells to prevent the generation of oxygen radicals on the lipid peroxidation, was compared with the untreated group as a control. After 1 h reperfusion at 37°C the hydroperoxide level obtained the liver without preservation in the untreated group was 12.4±2.4 nmol/100 mg lipid (n=4) and levels increased significantly by prolongation of the preservation time. On the other hand, the hydroperoxide level in the LEDD treated group did not change up to 24 h preservation. These results suggest that this improved assay for hydroperoxide level of PCOOH fraction in biological samples can be applied to investigations involving lipid peroxidation because of its simplicity and accuracy. Copyright (C) 2000 Elsevier Science B.V.

Original languageEnglish
Pages (from-to)515-520
Number of pages6
JournalJournal of Pharmaceutical and Biomedical Analysis
Volume23
Issue number2-3
DOIs
Publication statusPublished - 2000 Aug 15

Keywords

  • 1-Hexadecanoyl-2-[9, or 10-hydroperoxyl octadecanoyl]-sn-glycero-3-phosphocholine
  • 2-Methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo[1,2-a]pyrazin-3-one
  • Chemiluminescent assay
  • Phosphatidylcholine hydroperoxide

ASJC Scopus subject areas

  • Analytical Chemistry
  • Pharmaceutical Science
  • Drug Discovery
  • Spectroscopy
  • Clinical Biochemistry

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