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

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 tC 18 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.