Engelsk titel: Cell based methods for measuring lipid peroxidation and antioxidant activities
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Författare:
Hofer, Tim
;
Olsen, Ragnar Ludvig
Email: tim.hofer@uit.no
Språk: Nor
Antal referenser: 19
Dokumenttyp:
Artikel
UI-nummer: 10113739
Sammanfattning
Background: Several medical conditions involve increased lipid peroxidation where free radicals damage cellular membranes. Unsaturated fatty acids oxidize in presence of oxygen after initiation by peroxides in presence of transition metals such as iron and copper. Intake of natural antioxidants, including metal chelators and radical scavangers, can decrease the rate of lipid peroxidation.
Materials and Methods: In order to study and identify novel antioxidants, we have established a liver cell (HepG2) based method in 96-well microplates, in which the lipid peroxidation rate is measured. The method, abbreviated CLPAA (Cellular Lipid Peroxidation Antioxidant Activity), is based on the cellmembrane soluble and highly oxidation sensitive red fluorescent probe C11-BODIPY. Lipid peroxidation is induced by adding lipophilic cumene hydroperoxide (cumOOH), and green fluorescent C11-BODIPY oxidation products are monitored using a plate reader.
Results and interpretation: The results show that measurement of the formation rate of green C11-BODIPY oxidation products works better than measuring losses of red non-oxidized C11-BODIPY. From dose-response tests of antioxidants over broad concentration ranges, half maximal inhibitory concentrations (called IC50-values) are calculated for comparison of antioxidant efficiency. The method is demonstrated with direct comparison of the iron-binder desferrioxamine (DFO) with the flavonoid luteolin, capable of both binding metals and neutralizing free radicals. Following one hour of incubation, luteolin offered greater protective antioxidant effect (IC50 = 5,21 μM) than DFO (IC50 = 784 μM).