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

A chemiluminescent assay for hydroperoxide level of phosphatidylcholine hyd roperoxide (PCOOH) fraction purified from biological samples was presented. This method utilized of two Sep-Pak cartridges. A lipid soluble fraction w as isolated from each homogenized tissue or blood by Folch's method. The mi xture of phosphatidylcholine (PC) and PCOOH was separated from the lipid so luble fraction by a Sep-Pak silica cartridge. A Sep-Pak tC(18) cartridge ma de complete separation of both PCOOH and PC possible. The hydroperoxide lev el 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 ch emiluminescent dye. The mixture of positional isomers, 1-hexadecanoyl-2-[9, or 10-hydroperoxyl octadecanoyl]-sn-glycero-3-phosphocholine was used as a n 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 3.5 to 20 nmol, a nd the detection limit of the standard was 10 pmol (signal-ro-noise ratio > 3). This method was applied to the investigation of the lipid peroxidation induced by reperfusion of the liver with cold preservation, mimicking live r transplantation in rats. The effect of liposome-encapsulated dichlorometh ylene 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 degrees C th e hydroperoxide level obtained the liver without preservation in the untrea ted group was 12.4 +/- 2.4 nmol/100 mg lipid (n = 4) and levels increased s ignificantly by prolongation of the preservation time. On the other hand, t he hydroperoxide level in the LEDD treated group did not change up to 24 h preservation. These results suggest that this improved assay for hydroperox ide level of PCOOH fraction in biological samples can be applied to investi gations involving lipid peroxidation because of its simplicity and accuracy . (C) 2000 Elsevier Science B.V. All rights reserved.