CHARACTERIZATION OF LIPID HYDROPEROXIDES GENERATED BY PHOTODYNAMIC TREATMENT OF LEUKEMIA-CELLS

A new technique, high-performance liquid chromatography with reductive mode electrochemical detection on a mercury drop (HPLC-EC), has been used for analyzing lipid hydroperoxide (LOOH) formation in photooxidat ively stressed L1210 leukemia cells. Highly specific and sensitive for peroxides (detection limits <0.5 pmol for cholesterol hydroperoxides and <50 pmol for phospholipid hydroperoxides), this approach allows di fferent classes of LOOH to be separated and determined in minimally da maged cells. L1210 cells in serum containing growth medium were irradi ated in the presence of merocyanine 540 (MC540), a lipophilic photosen sitizing dye. Lipid extracts from cells exposed to a light fluence of 0.11 J/cm(2) (which reduced clonally assessed survival by 30%) showed 12-15 well-defined peaks in HPLC-EC. None of these peaks was observed when cells were irradiated without MC540 or when dye/light-treated sam ples were reduced with triphenylphosphine prior to analysis. Three pea ks of relatively low retention time (<12 min) were assigned to the fol lowing species by virtue of comigration with authentic standards: 3 be ta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide (5 alpha-OOH), 3 beta -hydroxycholest-4-ene-6 beta-hydroperoxide (6 beta-OOH), and 3 beta-hy droxycholest-5-ene-7 alpha/7 beta-hydroperoxide (7 alpha/7 beta-OOH). Formation of 5 alpha-OOH and 6 beta-OOH (singlet oxygen adducts) was c onfirmed by subjecting [C-14]cholesterol-labeled cells to relatively h igh levels of photooxidation and analyzing extracted lipids by HPLC wi th radiochemical detection. Material represented in a major peak at 18 -22 min on HPLC-EC was isolated in relatively large amounts by semipre parative HPLC and shown to contain phospholipid hydroperoxides (predom inantly phosphatidylcholine species, PCOOH) according to the following criteria: (i) decay of 18-22 min peak during Ca2+/phospholipase A(2) treatment, with reciprocal appearance of fatty acid hydroperoxides; (i i) reduction of peroxide during treatment with reduced glutathione and phospholipid hydroperoxide glutathione peroxidase, but not glutathion e peroxidase; and (iii) comigration with PCOOH standards in thin-layer chromatography. HPLC-EC analysis revealed quantifiable amounts of PCO OH and ChOOH at a light fluence that clonally inactivated <10% of the cells, which allows for the possibility that photoperoxidative damage plays a causal role in cell killing.