A Protocol for Quantifying Lipid Peroxidation in Cellular Systems by F2-Isoprostane Analysis

Cellular systems are essential model systems to study reactive oxygen species and oxidative damage but there are widely accepted technical difficulties with available methods for quantifying endogenous oxidative damage in these systems. Here we present a stable isotope dilution UPLC-MS/MS protocol for measuring F2-isoprostanes as accurate markers for endogenous oxidative damage in cellular systems. F2-isoprostanes are chemically stable prostaglandin-like lipid peroxidation products of arachidonic acid, the predominant polyunsaturated fatty acid in mammalian cells. This approach is rapid and highly sensitive, allowing for the absolute quantification of endogenous lipid peroxidation in as little as ten thousand cells as well as damage originating from multiple ROS sources. Furthermore, differences in the endogenous cellular redox state induced by transcriptional regulation of ROS scavenging enzymes were detected by following this protocol. Finally we showed that the F2-isoprostane 5-iPF2α-VI is a metabolically stable end product, which is excreted from cells. Overall, this protocol enables accurate, specific and sensitive quantification of endogenous lipid peroxidation in cellular systems.


Introduction
Reactive oxygen species (ROS) are formed during normal cellular metabolism and it is becoming increasingly apparent that they have an important, yet complicated role in biology and pathology [1,2]. The mitochondria are the main source of ROS production during the process of oxidative phosphorylation [3]. Other sources of ROS include fatty acid oxidation in the peroxisomes and enzyme complexes like NADPH oxidase [4]. Superoxide (•O 2 -), the primary ROS, can be further reduced to hydrogen peroxide (H 2 O 2 ) and the hydroxyl-radical (OH •-) in the cell. As a result of the high reactivity of these molecules, they readily react with DNA, proteins and lipids to cause oxidative damage, thereby altering their function towards pathology. In addition to toxicity, it has recently become clear that ROS and even ROS damage can act as secondary messengers in signal transduction in important metabolic pathways [5][6][7].
To control ROS, cells are equipped with specific defence and repair mechanisms to ensure cellular survival. Superoxide dismutases (SOD) are the main scavengers of •O 2 -, reducing it to H 2 O 2 , which in turn is scavenged by catalases and glutathione peroxidases. Peroxiredoxins are important organic hydroperoxide scavengers and play a crucial role in redox signalling [8,9]. ROS are also scavenged by small molecules, including GSH, ascorbic acid and α-tocopherol [8] The most challenging aspect of studying ROS metabolism is the fact that they are extremely reactive and short-lived molecules, which make them difficult to measure. Various approaches have been developed for cell-based systems to measure ROS and ROS damage, but many suffer from a lack of specificity, linearity or detailed method characterization.
Isoprostanes are chemically stable prostaglandin-like lipid peroxidation products that are endogenously formed from oxidative damage to polyunsaturated fatty acids (PUFA's) [10]. These molecules have been used as markers for oxidative stress in human pathologies and are considered to be the gold standard in measuring systemic lipid peroxidation in mammalian plasma and urine [11]. Recently, F3-isoprostanes have been identified [12] and were used as sensitive endogenous markers of oxidative damage in C. elegans [2]. Although various GC-and LC-MS methods exist for F2isoprostane measurement, these are all optimized for analysis in mammalian plasma and/or urine and do not address F2isoprostane measurement in cellular systems.
Here we characterize a liquid chromatography mass spectrometry isotope dilution based protocol optimized to quantify F2-isoprostanes in cellular systems and show that they are reliable markers of lipid peroxidation from various ROS sources. The developed protocol is rapid, lacks derivatization and is highly sensitive and linear over a wide dynamic range, allowing for absolute quantification of endogenous lipid peroxidation in as little as ten thousand cells. Furthermore, although some F2-isoprostanes have been shown to be further metabolized, we demonstrate that the F2-isoprostane 5-iPF 2α -VI is highly stable and excreted from cells. Finally, this F2isoprostane approach showed high sensitivity, which allowed for the assessment of the endogenous redox changes caused by activation of a Forkhead transcription factor, which regulates transcription of ROS scavenging enzymes. This protocol for measuring cellular lipid peroxidation by F2-isoprostane analysis may be an important tool to contribute to a better understanding of ROS metabolism in cell based systems.

Cell culture and pro-oxidant treatment
The human hepatocellular carcinoma (HepG2) cell line and the human colon carcinoma cell line DLD-1 were both obtained from ATCC and DL-23 cells were generated from the DLD-1 line as described previously [13]. All cell lines were cultured at 37°C under a humidified 5% CO 2 atmosphere in DMEM + GlutaMax containing 4.5 g/L glucose (Gibco), supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Gibco). Experiments were performed in 9 cm tissue culture plates containing 10 ml of culture media. Pro-and anti-oxidants were added to media and incubated for indicated times before harvesting.

Sample preparation
Cells were harvested in ice-cold PBS and collected in a 1,5 ml centrifuge tube at 4°C and spiked with 2 ng deuterated internal standards before homogenization. Samples were homogenized using the Bullet Blender® (NextAdvance) with zirconium(VI)oxide (ZrO 2 ) beads (0.5 mm), according to manufacturer's instructions at 4°C. Homogenates were then hydrolyzed in 3 M KOH, containing 100 µM BHT [14] (3:1 v:v H 2 O:MeOH) for 45 min. at 45°C. Next, samples were cooled to room temperature and acidified to pH < 3 with HCl. Samples were then transferred to glass tubes and isoprostanes were extracted by vortexing with 5 ml ethyl acetate for 1 min., subsequently equilibrated on ice for 5 min., centrifuged for 5 min. at 3500g, 4°C, saving the organic phase for further solid phase extraction (SPE). NH 2 Sep-Pak cartridges (Waters) were preconditioned with 5 ml hexane and subjected to a vacuum to obtain a flow rate of approximately 3 ml/min. An equal volume of ethyl acetate was used to rinse before elution with 3 ml ethyl acetate: MeOH: acetic acid (10:85:5 v:v:v). Eluates were dried under nitrogen and re-suspended in 150 µl 0.15% NH 4 OH: acetonitrile (90:10 v:v).

Ultra-high performance liquid chromatography tandem mass spectrometry
Liquid chromatography was performed on an ACQUITY UPLC® system with a binary solvent manager (Waters). Isoprostanes were separated using an analytical BEH UPLC C18 column (150 mm x 2.1 mm, 1.7 µm particle size, Waters) at 40°C, over a linear gradient from 10-30% solvent B (solvent A= 0.15% NH 4 OH, solvent B= 95% acetonitrile: 5% MeOH, 0.0125% NH 4 OH) in 15 min. Column eluate was directly coupled to a Xevo™ TQ Mass spectrometer (Waters) fitted with an electron spray ionization probe operating in the negative ion mode with argon as collision gas. Cone voltage was set at 35 V, capillary voltage at 2.4 kV and desolvation gas temperature at 600°C with a flow rate of 750 (l/hrs). Multiple reaction monitoring (MRM) was used to analyze the various isoprostanes. F2-class VI isoprostanes were measured with a transition m/z 353>115 and F2-class III with transition m/z 353>193. Deuterated F2-class VI and III isoprostanes were measured with transitions m/z 362>115 and 357>197 respectively. Isoprostane concentrations relative to their labeled internal standards were determined using TargetLynx TM (Waters) software. For each analysis, a fresh six point concentration series was prepared.

In vitro peroxidation assay
Arachidonyl PAF-C16 (Cayman Chemicals) was stored as a 5 mM stock solution in 100% ethanol under nitrogen at -20°C to ensure minimal auto-oxidation. For analysis, arachidonyl PAF-C16 was diluted to 5 μM in PBS, containing 50 μM CuCl 2 and incubated at 37°C for indicated times in the presence or absence of 100 µM BHT. HepG2 cell homogenates were incubated for 7 hours at 37°C in the presence of CuCl 2 (50 μM), either with or without BHT or Trolox (10 mM) followed by extraction and MS analysis.

Quantification and statistical analysis
All data were normalized to the amount of protein per sample, using Bradford (BioRad) analysis according to manufacturer's instruction. Experiments were performed in triplicate unless stated otherwise. Student t-test was used to test for significant differences between groups at p < 0.05.

F2-isoprostanes are formed from peroxidation of arachidonic acid
Arachidonic acid (AA) is a C20:4 omega-6 fatty acid and is the predominant polyunsaturated fatty acid (PUFA) in mammalian cells. It plays an important part in maintaining cell membrane integrity, fluidity and rigidity. It also acts as a precursor for eicosanoids, which are important signaling molecules. AA contains four double bonds, rendering it sensitive to ROS damage, which results in the formation of four F2-isoprostane isomer classes ( Figure 1A). Theoretically, each class can exist of 8 diasteriomeric forms for a total of 64 distinct isomers [15]. F2-isoprostanes are formed from AA esterified to phospholipids and to confirm this, we subjected arachidonyl PAF-C16, a glycerol-3-phosphocholine that contains AA to in vitro copper-induced lipid peroxidation and monitored F2isoprostane formation using mass spectrometry. Copperinduced lipid peroxidation of arachidonyl PAF-C16 leads to the formation of lipid peroxyl radicals which initiate a chain of lipid peroxidation reactions resulting in F2-isoprostane formation [2,16,17].
Multiple Reaction Monitoring (MRM) was used to achieve optimum selectivity for the mass spectrometry analysis of the F2-isoprostanes. MRM monitors the precursor ion masses and, after fragmentation, the most predominant product ion masses. MRM transitions were determined by performing product ion scans for the F2-class-III and F2-class-VI isoprostanes ( Figure  1B). The MRM pair identified for endogenous 8-isoPGF2α-III was m/z 353>193 while it was m/z 353>115 for both 5-iPF 2α -VI and 8,12-iso-iPF 2α -VI. Although F2-class-VI isoprostanes have an identical MRM pair, they can be separated based on their unique retention times ( Figure S1). Deuterated internal standards were used for absolute quantification. They have the same chemical properties and therefore the same retention times as endogenous F2-isoprostanes but with a higher m/z value, allowing for specific detection of F2-isoprostanes by coelution. Their MRM pairs were determined in the same way as that of endogenous F2-isoprostanes and were found to be m/z 357>197 for 8-isoPGF2α-III-d4 and m/z 364>115 for both 5-iPF 2α -VI-d11 and 8,12-iso-iPF 2α -VI-d11. By using these MRM transitions in combination with their chromatographic retention and co-elution with their corresponding deuterated internal standards, we demonstrated the formation of three F2isoprostane isomers including 8-isoPGF 2α -III; 5-iPF 2α -VI and 8,12-iso-iPF 2α -VI upon in vitro peroxidation of AA (Figure 2A-C). Note that an alternative isoprostane nomenclature is also in use [18]. These isomers were chosen since they are abundantly formed and synthetic standards are commercially available. There was a clear increase in the levels of all three isomers over time in response to copper treatment (Figure 2A-C). This increase was blunted by the presence of butylated hydroxytoluene (BHT), an antioxidant that is known to terminate lipid peroxidation. It is interesting to note that all three isomers behave similarly in response to copper although, 8-isoPGF 2α -III (Figure 2A) showed the highest increase and 8,12iso-iPF 2α -VI levels ( Figure 2C) showed a lower response. To determine if these F2-isoprostanes are also formed in cells, lysates from the human liver carcinoma HepG2 cell line were prepared and subjected to copper-induced in vitro lipid peroxidation. Again, a clear response to the copper treatment for all the isomers was noted (Figure 2D-F), whereas in the presence of BHT and Trolox, a water-soluble vitamin E derivative, there was no increase in F2-isoprostane levels ( Figure 2D-F). Next, isoprostane formation in living cells was tested by treating HepG2 cells with Glucose oxidase (GOX), which generates a constant flux of H 2 O 2 in the presence of glucose and oxygen. Figure 2G shows the overlay of the chromatograms of extracted F2-isoprostanes from cells treated for indicated times. It is clear that the areas under several peaks increased with increasing exposure time to GOX. These findings suggest that these peaks belong to endogenous F2isoprostanes, which was confirmed by inclusion of the deuterated internal standards that were spiked to the sample after GOX treatment. Several peaks however that did respond to GOX, did not co-elute with the deuterated internal standards. Although it is possible that these belong to endogenous F2isoprostane isomers, the absence of internal standards currently prevent definitive identification. Therefore, peroxidation of arachidonyl PAF-C16, cell lysates and living cells resulted in the formation of at least three F2-isoprostane isomers as markers of lipid peroxidation.

Method characterization, F2-isoprostane stability and metabolic fate in cells
To characterize this protocol, synthetic and deuterated internal standards of two different F2-isoprostane isomers were used. Cell lysates are extremely complex as metabolite mixtures containing multiple molecules that potentially influence the extraction procedure of the isoprostanes as well as causing ion suppression in the source of the mass spectrometer, thereby affecting the analysis of F2isoprostanes. We tested a range of extraction methods to optimize the protocol for cells and found that a combination of liquid/liquid extraction (LLE) and solid phase extraction (SPE) with aminopropyl (NH 2 ) as stationary phase was optimal for F2isoprostane detection in cells (Fig. 3). A detailed description of this protocol can be found in the Materials and Methods section. To demonstrate that there is no matrix affect after extraction with this protocol, a series of cell lysates and PBS blanks were spiked with increasing concentrations of 8-isoPGF 2α -III and 5-iPF 2α -VI to compare the slopes of the fitted curves (Fig. 4A). We show similar slopes of the curves for the two isomers and identified a linear response over a ten thousand fold dynamic range (Fig. 4A). This finding confirmed that there is no effect of the metabolite matrix in cell lysates on extraction and ionization efficiency for the MS analysis of F2isoprostanes using this protocol. Next, a cell number titration experiment was performed to establish the lower limit of the number of cells by which lipid peroxidation can be quantified. Using this protocol, 5-iPF 2α -VI could be measured in as little as ten thousand cells with a signal to noise ratio (S/N) of 30 (Figures S2A and S2B). Furthermore, the limit of quantification (LOQ), calculated as a S/N of 10, was determined to be 1.5 pg/ml and the limit of detection set at S/N of 3 was 500 fg/ml. To the best of our knowledge, these are the lowest reported  levels thus far. These findings suggest that the F2-isoprostane measurement is highly sensitive, allowing for the endogenous quantification of lipid peroxidation in as little as ten thousand HepG2 cells. Together, these results show that this UPLC-MS/MS stable isotope dilution assay, optimized for extracting and measuring F2-isoprostanes in cellular systems, allows for the detection and quantification of F2-isoprostanes in a robust and sensitive manner. The approach is furthermore linear over a wide dynamic range and, by making use of the deuterated internal standards, absolute quantification of F2-isoprostanes is possible.
One of the caveats of measuring oxidative damage is the fact that there is a constant turnover and repair of damaged molecules and these molecules might be chemically or metabolically unstable. In order to address this question for F2isoprostanes, we spiked a HepG2 cell lysate with deuterated standards of 8-isoPGF 2α -III and 5-iPF 2α -VI, followed by longitudinal incubation at 25°C and 37°C to assess their stability under physiological conditions. We found that over a period of 56 hours there were no significant changes in the levels of the added deuterated standards for both isomers ( Figure 4B). This indicates that these two F2-isoprostane isomers are highly stable. To further determine the metabolic fate of the F2-isoprostanes we subjected HepG2 cells to 2 hours of GOX treatment to induce F2-isoprostane formation and monitored these levels in time. A threefold increase in F2isoprostane levels was observed ( Figure 4C) and after removal of GOX from the medium, there was no significant decrease in F2-isoprostane levels in the cell lysate or an increase in excreted isoprostane levels in the culture media within the first 4 hours. (Figure 4C and 4D). However, after 24 hours cellular F2-isoprostane levels were restored to that of baseline levels ( Figure 4C) and excretion was observed in the culture media ( Figure 4D). In contrast, the kinetics of the 8-isoPGF 2α -III isomer were different. Already 1 hour after GOX removal, 8-isoPGF 2α -III levels turned to baseline and excretion occurred more rapidly than for the 5-iPF 2α -VI isomer, albeit at much lower levels (Figure 4E and 4F). These findings suggest that 5-   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,). iPF 2α -VI is more stable than the 8-isoPGF 2α -III isomer in agreement with the notion that 8-isoPGF 2α -III has been observed to be biologically active and subject to β-oxidation [15]. Therefore, we continued our analysis with the 5-iPF 2α -VI isomer.

F2-isoprostanes are formed in response to different ROS sources
To further explore the use of F2-isoprostanes as markers of oxidative damage in cellular systems, we subjected HepG2 cells to various pro-and anti-oxidants. Paraquat generates superoxide through redox cycling after take up by the cell, using NADPH as electron donor [19]. Cells were treated with different concentrations of PQ for 24 hours at 37°C and F2isoprostane levels were measured. In cells exposed to concentrations lower than 100 µM PQ, no changes in 5-iPF 2α -VI levels were observed. However, a significant increase in 5-iPF 2α -VI levels was measured in cells treated with PQ concentrations equal to or above 100 µM and a more than 2 fold increase was observed after treatment with 500 µM compared to vehicle treated cells. (Figure 5A). In contrast, when stimulated with 500 µM PQ in the presence of Trolox, there was no increase in 5-iPF 2α -VI levels ( Figure S3). These results show that PQ induced detectable cellular oxidative damage after treatment for 24 hours at concentrations ≥ 100 µM. Next, we subjected cells to an exogenous source of H 2 O 2 by means of GOX treatment. Cells were harvested every 20 minutes for 2 hours and analyzed for levels of lipid peroxidation. There was a linear increase in 5-iPF 2α -VI levels over time with a maximum response of about 3-fold induction ( Figure 5B). This suggests that lipid peroxidation increases over time in the presence of H 2 O 2 in an accumulative manner. Since the mitochondria are considered as the main source of intracellular ROS, we treated cells with rotenone, a compound that generates ROS in the mitochondria by inhibiting electron flow in complex I in the electron transport chain (ETC). Rotenone treatment for 24 hours caused a dose dependent increase in 5-iPF 2α -VI levels ( Figure 5C). This confirms that a blockage of electron flow in the ETC leads to an increase in ROS and ultimately to an increase in lipid peroxidation. The hydroxyl-radical (OH •-) is another critical ROS and is known to cause damage indiscriminately to DNA, proteins and lipids. The free radical generator 2,2'-azobis-2-methyl-propanimidamine dihydrochloride (AAPH) constantly produces OH •-in solution during its decomposition [20]. A 2-fold increase in 5-iPF 2α -VI levels was observed after 24 hours of AAPH treatment ( Figure  5D). Treatment with AAPH in the presence of Trolox did not result in an increase in lipid peroxidation. It is interesting to note that the control cells, treated with Trolox, in the absence of any ROS generator showed decreased 5-iPF 2α -VI levels as compared to the untreated controls ( Figure 5D). This suggests that Trolox blocks endogenous ROS damage. Taken together, these results indicate that F2-isoprostanes are sensitive markers of cellular lipid peroxidation in response to multiple ROS.

FOXO3a increase the resistance of cells against oxidative stress
Forkhead box O (FOXO) transcription factors are important regulators of cellular defense against ROS by controlling the gene transcription of ROS-scavenging enzymes, including SODs and catalases [21]. We employed two cell lines, DL-23 and DLD-1 to test the role of FOXO transcription of the cellular oxidative damage formation. The human colon carcinoma DL-23 cell line stably expresses a constitutively active FOXO3a construct fused to a modified form of the estrogen receptor hormone-binding domain which allows conditional activation of FOXO3a [22]. By adding the synthetic estrogen receptor agonist 4-hydroxy tamoxifen (4-OHT), the fusion protein is activated by entering the nucleus to initiate transcription [22]. The DLD-1 parental cell line, which stably expresses the empty vector, was used as control. The effect of FOXO3a activation on the stress resistance after treatment with GOX and PQ was determined using these cell lines. Firstly, both DLD-1 and DL-23 cell lines were treated with 4-OHT for 16h, after which cells were treated with GOX for another 2 hours to induce oxidative stress. There was a significantly higher increase in F2-isoprostanes in the DLD-1 cells as compared to the DL-23 cells after GOX treatment, which suggests that cells with activated FOXO3a are more resistant to exogenous oxidative stress. Although there was a trend for lower baseline levels of lipid peroxidation in the activated DL-23 cells, this did not reach significance ( Figure 6A). To determine if a longer activation time of FOXO3a could significantly affect baseline levels of lipid peroxidation and protect against endogenously formed ROS, cells were stimulated for 24 hours before treating them with PQ for another 8 hours. In this case, there was indeed a significant decrease in baseline F2-isoprostane levels in the DL-23 cells 24 hours after FOXO3a activation. These findings suggest increased ROS scavenging or repair capacity due to prolonged FOXO3a activation. Furthermore, the DL-23 cells had less PQinduced oxidative damage as compared to the DLD-1 cells, indicating a FOXO3a-mediated protection against endogenous ROS damage ( Figure 6B). These results demonstrate that this UPLC-MS/MS protocol for measuring F2-isoprostanes in cells has the sensitivity to detect significant differences in the endogenous redox state controlled by transcription factor mediated scavenging capacity in cellular systems.

Discussion
With the discovery of F2-isoprostanes as noncyclooxygenase derived products of free radical-induced damage to AA-containing lipids [10], numerous studies showed them to be reliable markers of systemic oxidative damage in plasma and urine [15]. Here we present a protocol to accurately measure in vivo lipid peroxidation in cellular systems by using F2-isoprostane analysis. We show that at least three different F2-isoprostane isomers are formed in response to oxidative damage to an AA-containing phospholipid both in vitro and in living HepG2 cells. It is interesting to note that copper treatment induced the greatest response for the 8-isoPGF 2α -III isomer as compared to the other two monitored isomers in vitro (Figure 2). This observation is in agreement to the notion that there is a preference for the formation of different isomers during AA peroxidation [23]. However, when baseline levels of the F2-isoprostanes were measured in cultured cells 8-isoPGF 2α -III levels were remarkably found to show the lowest concentration (Figure 4). It is possible that 8-isoPGF 2α -III is further metabolized in the cell, as rapid clearance and excretion was observed (Figure 4). This is in accordance with studies which have shown that 8-isoPGF 2α -III  is subject to β-oxidation [15]. The isomer 5-iPF 2α -VI however, showed a good response to in vitro peroxidation and baseline levels in cells were relatively high. No evidence thus far has been presented that shows that this isomer is further metabolized. This is in agreement with the high stability and efficient clearance that was observed (Figure 4). Therefore, we propose to use this isoform as marker for oxidative damage in cellular systems.
By using deuterated internal standard we were able to fully optimize and characterize this method in cells. By comparing the curve of an increasing concentration of F2-isoprostanes spiked in PBS and cell lysates, we could determine that the matrix had no effect on this extraction protocol of F2isoprostanes and showed no ion suppression during mass spectrometry. We show that this method has a high dynamic range of ten thousand fold. It is extremely sensitive and we can measure 5-iPF 2α -VI in as little as ten thousand cells with high confidence. Several technical papers have been published in previous years that describe isoprostane analysis by GC-and LC-MS. Although GC-MS technology is known to yield reproducible, robust and sensitive detection and quantification, the technology is limited in terms of throughput as it required time-consuming sample preparation and derivatization protocols [24]. LC-MS methods on the other hand are less time consuming but struggle to achieve GC-MS-like sensitivity [25,26]. Our protocol employs UPLC-MS/MS and through our optimized sample preparation procedure, high throughput studies to measure lipid peroxidation in cells during screening of compound libraries in the pharmaceutical industry are feasible. In fact, the lower limit of quantification reported here is the most sensitive reported of any GC-or LC-MS method to date.
To validate this protocol, we subjected cells to different proand anti-oxidants and assessed oxidative damage through F2isoprostane measurement. Levels of isoprostanes increased in response to different ROS species from different sources, including H 2 O 2 , OH •-and •O 2 -. Although H 2 O 2 is not a radical by itself, a Fenton reaction is induced in the presence of transition metal ions, which catalyze hydroxyl radical formation. As expected, there was a dose-dependent increase in oxidative damage in response to rotenone treatment. This demonstrated that this approach can assess damage originating from the mitochondria and more specifically from oxidative phosphorylation, which is considered the main source of baseline ROS and ROS damage in the cell. Interestingly, similar to rotenone, PQ also produces superoxide radicals, yet the kinetics of F2-isoprostane formation differed (Figure 5). These findings might suggest that oxidative damage formation from superoxide produced by PQ treatment is distinct from the mitochondria. An attractive hypothesis would be that PQ mediates superoxide induction predominantly localized to cytoplasmic compartments. In line with this notion, PQ treatment was recently found to strongly induce oxidative damage in the nematode C. elegans, deficient in cytoplasmic SOD activity. In contrast, nematodes deficient in mitochondrial dismutase activity showed little response to PQ, which otherwise responded strongly to rotenone [2]. Further kinetic studies will be required to study the superoxide formation by PQ in detail.
The Forkhead box O transcription factors FOXO are important, conserved players in regulating cellular defense and repair mechanisms through transcriptional control of genes involved in ROS scavenging and DNA repair [27]. In addition to the study of the redox balance in cells in response to direct activators or inhibitors of ROS, an indirect system for redox analysis was used to test the sensitivity of the approach. By using an inducible FOXO3a expressing cell line, the endogenous redox response of transcription factor mediated redox control could be monitored, indicating that the F2iP protocol is sensitive enough to detect indirect redox control.
Taken together, the 5-iPF 2α -VI F2-isoprostane isomer is a stable marker for sensitive, accurate and linear quantification of endogenous lipid peroxidation in cellular systems, which could prove valuable for studies into the role of ROS and its damage in cells. This straightforward and short sample preparation protocol furthermore should allow high-throughput analysis. Figure S1. A representative chromatogram of the MRM transition of F2-Class-VI isoprostanes demonstrating the chromatic separation of 5-iPF 2α -VI and 8,12-iso-iPF 2α -VI and co-elution of deuterated internal standards 5-iPF 2α -VI-d11 and 8,12-iso-iPF 2α -VI-d11.