Figure 1.
The chemical structure and mass spectrum of F2-isoprostanes.
(A) Four isomeric classes of F2-isoprostanes are formed as a result of oxidative damage to arachidonic acid. (B) The product ion scan of F2-class-III and F2-class-VI isoprostanes. The ion masses indicated in red were used as MRM transition pairs for the indicated isoprostanes.
Figure 2.
F2-isoprostanes are formed from peroxidation of arachidonic acid.
(A-C) Copper-induced in vitro peroxidation of arachidonic acid resulted in the formation of three F2-isoprostane isomers. Arachidonyl PAF-C16 was subjected to 50 µM of CuCl2, with or without BHT, for the indicated time points and the levels of 8-isoPGF2α-III; 5-iPF2α-VI and 8,12-iso-iPF2α-VI were measured by UPLC-MS/MS. (D-F) F2-isoprostanes were formed in a time-dependent manner in cell lysates upon copper induced lipid peroxidation. HepG2 cell lysates were incubated with CuCl2 for indicated time points in the presence or absence of Trolox or BHT. Levels of 8-isoPGF2α-III; 5-iPF2α-VI and 8,12-iso-iPF2α-VI were analyzed in time by UPLC-MS/MS. Data are represented as mean ± S.D. (G) UPLC-MS/MS chromatogram overlay of extracted F2-isoprostanes of cells treated with GOX for indicated times.
Figure 3.
A schematic flow diagram of the sample preparation protocol for F2-isoprostane extraction from cultured cells.
For detailed description, see materials and methods section. Abbreviations are: ZrO2 (zirconium(VI)oxide); RT (room temperature); LLE (liquid/liquid extraction); SPE (solid phase extraction).
Figure 4.
Characteristics of F2-isoprostane analysis in cells.
(A) 8-isoPGF2α-III and 5-iPF2α-VI quantification is not affected by ion suppression. UPLC-MS/MS intensities of increasing concentrations of 8-isoPGF2α-III and 5-iPF2α-VI standards in PBS or cell lysates relative to a fixed amount (2ng) of their respective deuterated internal standard were plotted. (B) F2-isoprostanes are stable in physiological conditions. 8-isoPGF2α-III-d4 and 5-iPF2α-VI-d11 were spiked into a HepG2 cell lysate and incubated at 25 or 37°C for indicated time points. F2-isoprostanes were measured as in Figure 4A and scatter plots show individual data points. (C-F) 8-isoPGF2α-III and 5-iPF2α-VI are excreted in the culture media at different rates by HepG2 cells. HepG2 cells were treated with GOX for 2 hours to induce oxidative damage where after GOX was removed and cells were allowed to recover for indicated times. F2-isoprostane levels were monitored as in Figure 4A. Results are shown as mean fold increase ± S.D (* p < 0.05, ** p < 0.01,).
Figure 5.
F2-isoprostanes are formed in response to different ROS sources.
(A) F2-isoprostane levels increased in response to PQ treatment. HepG2 cells were treated with increasing concentrations of PQ for 24 hours. 5-iPF2α-VI levels were measured by UPLC-MS/MS. (B) Exogenous H2O2 exposure of HepG2 cells resulted in a 3-fold increase in levels of 5-iPF2α-VI. HepG2 cells were incubated in the presence of GOX for 2 hours and 5-iPF2α-VI levels were analyzed every 20 minutes. (C) Rotenone increased lipid peroxidation in HepG2 cells in a dose-dependent manner. HepG2 cells were treated with an increasing concentration of rotenone and F2-isoprostanes were measured after 24 hours. (D) Lipid peroxidation induced by the peroxyl radical generator, AAPH, is quenched by the antioxidant Trolox. Cells were treated with either Trolox, AAPH or both, for 24 hours. Data are shown as mean fold increase ± S.D over untreated control cells (** p < 0.01, *** p < 0.001).
Figure 6.
The F2-isoprostane approach allows for analysis of FOXO transcription factor control of endogenous oxidative stress.
(A) GOX-induced lipid peroxidation is significantly decreased when FOXO3a is activated. DLD-1 and DL-23 cells were treated with 4-OHT to activate FOXO3a for 16 hours before H2O2 exposure by GOX treatment for 2 hours. F2-isoprostane levels were analyzed by UPLC-MS/MS and are shown as mean fold increase ± S.D (* p < 0.05) (B) Prolonged FOXO3a activation increased the protection against endogenously formed ROS damage in colon carcinoma cells. DLD-1 and DL-23 cells were incubated with 4-OHT to activate FOXO3a for 24 hours, before treatment with PQ for another 8 hours. 5-iPF2α-VI levels were analyzed and data are represented as mean fold increase ± S.E.M (*p < 0.05, *** p < 0.001).