LC-HRMS analysis of phospholipids bearing oxylipins.

BACKGROUND: Several oxylipins including hydroxy- and epoxy-polyunsaturated fatty acids act as lipid mediators. In biological samples they can be present as non-esterified form, but the major part occurs esterified in phospholipids (PL) or other lipids. Esterified oxylipins are quantified indirectly after alkaline hydrolysis as non-esterified oxylipins. However, in this indirect analysis the information in which lipid class oxylipins are bound is lost. In this work, an untargeted liquid chromatography high-resolution mass spectrometry (LC-HRMS) method for the direct analysis of PL bearing oxylipins was developed. RESULTS: Optimized reversed-phase LC separation achieved a sufficient separation of isobaric and isomeric PL from different lipid classes bearing oxylipin positional isomers. Individual PL species bearing oxylipins were identified based on retention time, precursor ion and characteristic product ions. The bound oxylipin could be characterized based on product ions resulting from the α-cleavage occurring at the hydroxy/epoxy group. PL sn-1/sn-2 isomers were identified based on the neutral loss of the fatty acyl in the sn-2 position. A total of 422 individual oxPL species from 7 different lipid classes i.e., PI, PS, PC, PE, PC-P, PC-O, and PE-P were detected in human serum and cells. This method enabled to determine in which PL class supplemented oxylipins are incorporated in HEK293 cells: 20:4;15OH, 20:4;14Ep, and 20:5;14Ep were mostly bound to PI. 20:4;8Ep and 20:5;8Ep were esterified to PC and PE while other oxylipins were mainly found in PC. SIGNIFICANCE: The developed LC-HRMS method enables the comprehensive detection as well as the semi-quantification of isobaric and isomeric PL species bearing oxylipins. With this method, we show that the position of the oxidation has a great impact and directs the incorporation of oxylipins into the different PL classes in human cells.

Plasma levels of EPA and DHA after ingestion of a single dose of EPA and DHA ethyl esters.

Omega-3 polyunsaturated fatty acids (n3 PUFA), specifically eicosapentaenoic acid (EPA, 20:5n3), and docosahexaenoic acid (DHA, 22:6n3), are essential for maintaining health. To better understand their biology, it is important to define their bioavailability. The aim of this cross-over study was to investigate and compare the acute effects on plasma EPA and DHA levels after single doses of EPA oil (99% pure) and DHA (97% pure) ethyl esters. Twelve men aged 20-40 years with a body-mass-index of 20-27 kg/m2 and low fish consumption were recruited. Several measures (e.g., 4-week run-in period, standardized diet, and blood collection protocols) were taken to reduce the inter-individual variability of plasma fatty acids levels. Using a cross-over design, the subjects received 2.2 g of EPA in the first test period and 2.3 g of DHA in the second. The test periods were separated by 2 weeks. Blood samples were taken before dosing and after 2, 4, 6, 8, 12, 24, 48, and 72 h. The mean ± SE maximum concentrations for EPA were higher than for DHA (115 ± 11 µg/mL vs. 86 ± 12 µg/mL; p = 0.05). The mean ± SE incremented area under the plasma concentration curve over 72 h for EPA (2461 ± 279 µg/mL) was 2.4 times higher (p < 0.001) than that for DHA (1021 ± 170 µg/mL). The mean ± SE half-life was for EPA and DHA was 45 ± 8 and 66 ± 12 h. Our results indicate that EPA administration in single doses leads to higher circulating plasma levels of EPA compared to an effect of an equivalent dose of DHA on DHA plasma levels.

Stearidonic acid improves eicosapentaenoic acid status: studies in humans and cultured hepatocytes.

Background: Ahiflower oil from the seeds of Buglossoides arvensis is rich in α-linolenic acid (ALA) and stearidonic acid (SDA). ALA and SDA are potential precursor fatty acids for the endogenous synthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are n3-long chain polyunsaturated fatty acids (n3-LC-PUFAS), in humans. Since taurine, an amino sulfonic acid, is often associated with tissues rich in n3-LC-PUFAS (e.g., in fatty fish, human retina), taurine may play a role in EPA- and DHA-metabolism. Objective: To examine the capacity of the plant-derived precursor fatty acids (ALA and SDA) and of the potential fatty acid metabolism modulator taurine to increase n3-LC-PUFAS and their respective oxylipins in human plasma and cultivated hepatocytes (HepG2 cells). Methods: In a monocentric, randomized crossover study 29 healthy male volunteers received three sequential interventions, namely ahiflower oil (9 g/day), taurine (1.5 g/day) and ahiflower oil (9 g/day) + taurine (1.5 g/day) for 20 days. In addition, cultivated HepG2 cells were treated with isolated fatty acids ALA, SDA, EPA, DHA as well as taurine alone or together with SDA. Results: Oral ahiflower oil intake significantly improved plasma EPA levels (0.2 vs. 0.6% of total fatty acid methyl esters (FAMES)) in humans, whereas DHA levels were unaffected by treatments. EPA-levels in SDA-treated HepG2 cells were 65% higher (5.1 vs. 3.0% of total FAMES) than those in ALA-treated cells. Taurine did not affect fatty acid profiles in human plasma in vivo or in HepG2 cells in vitro. SDA-rich ahiflower oil and isolated SDA led to an increase in EPA-derived oxylipins in humans and in HepG2 cells, respectively. Conclusion: The consumption of ahiflower oil improves the circulating levels of EPA and EPA-derived oxylipins in humans. In cultivated hepatocytes, EPA and EPA-derived oxylipins are more effectively increased by SDA than ALA.

ALOX15B controls macrophage cholesterol homeostasis via lipid peroxidation, ERK1/2 and SREBP2.

Macrophage cholesterol homeostasis is crucial for health and disease and has been linked to the lipid-peroxidizing enzyme arachidonate 15-lipoxygenase type B (ALOX15B), albeit molecular mechanisms remain obscure. We performed global transcriptome and immunofluorescence analysis in ALOX15B-silenced primary human macrophages and observed a reduction of nuclear sterol regulatory element-binding protein (SREBP) 2, the master transcription factor of cellular cholesterol biosynthesis. Consequently, SREBP2-target gene expression was reduced as were the sterol biosynthetic intermediates desmosterol and lathosterol as well as 25- and 27-hydroxycholesterol. Mechanistically, suppression of ALOX15B reduced lipid peroxidation in primary human macrophages and thereby attenuated activation of mitogen-activated protein kinase ERK1/2, which lowered SREBP2 abundance and activity. Low nuclear SREBP2 rendered both, ALOX15B-silenced and ERK1/2-inhibited macrophages refractory to SREBP2 activation upon blocking the NPC intracellular cholesterol transporter 1. These studies suggest a regulatory mechanism controlling macrophage cholesterol homeostasis based on ALOX15B-mediated lipid peroxidation and concomitant ERK1/2 activation.

Sterol Derivatives Specifically Increase Anti-Inflammatory Oxylipin Formation in M2-like Macrophages by LXR-Mediated Induction of 15-LOX.

The understanding of the role of LXR in the regulation of macrophages during inflammation is emerging. Here, we show that LXR agonist T09 specifically increases 15-LOX abundance in primary human M2 macrophages. In time- and dose-dependent incubations with T09, an increase of 3-fold for ALOX15 and up to 15-fold for 15-LOX-derived oxylipins was observed. In addition, LXR activation has no or moderate effects on the abundance of macrophage marker proteins such as TLR2, TLR4, PPARγ, and IL-1RII, as well as surface markers (CD14, CD86, and CD163). Stimulation of M2-like macrophages with FXR and RXR agonists leads to moderate ALOX15 induction, probably due to side activity on LXR. Finally, desmosterol, 24(S),25-Ep cholesterol and 22(R)-OH cholesterol were identified as potent endogenous LXR ligands leading to an ALOX15 induction. LXR-mediated ALOX15 regulation is a new link between the two lipid mediator classes sterols, and oxylipins, possibly being an important tool in inflammatory regulation through anti-inflammatory oxylipins.

Changing from lipoprotein apheresis to evolocumab treatment lowers circulating levels of arachidonic acid and oxylipins.

Background and aims: Previous studies have shown that lipoprotein apheresis can modify the plasma lipidome and pro-inflammatory and pro-thrombotic lipid mediators. This has not been examined for treatment with protein convertase subtilisin/kexin type 9 inhibitors such as evolocumab, which are increasingly used instead of lipoprotein apheresis in treatment-resistant familial hypercholesterolemia. The aim of this study was to compare the effects of evolocumab treatment and lipoprotein apheresis on the fatty acid profile and on formation of lipid mediators in blood samples. Methods: We analyzed blood samples from 37 patients receiving either lipoprotein apheresis or evolocumab treatment as part of a previous study. Patients were stratified according to receiving lipoprotein apheresis (n = 19) and evolocumab treatment (n = 18). Serum fatty acid analysis was performed using gas chromatography flame ionization detection and plasma oxylipin analysis was done using liquid chromatography tandem mass spectrometry. Results: Changing from lipoprotein apheresis to evolocumab treatment led to lower levels of omega-6 polyunsaturated fatty acid (n-6 PUFA) including arachidonic acid, dihomo-γ-linolenic acid and linoleic acid. Moreover, several n-6 PUFA-derived oxylipins were reduced after evolocumab treatment. Conclusions: Given that arachidonic acid, either directly or as a precursor, is associated with the development of inflammation and atherosclerosis, evolocumab-mediated reductions of arachidonic acid and its metabolites might have an additional beneficial effect to lower cardiovascular risk.

LC-ESI-HRMS - lipidomics of phospholipids : Characterization of extraction, chromatography and detection parameters.

Lipids are a diverse class of molecules involved in many biological functions including cell signaling or cell membrane assembly. Owing to this relevance, LC-MS/MS-based lipidomics emerged as a major field in modern analytical chemistry. Here, we thoroughly characterized the influence of MS and LC settings - of a Q Exactive HF operated in Full MS/data-dependent MS2 TOP N acquisition mode - in order to optimize the semi-quantification of polar lipids. Optimization of MS-source settings improved the signal intensity by factor 3 compared to default settings. Polar lipids were separated on an ACQUITY Premier CSH C18 reversed-phase column (100 × 2.1 mm, 1.7 µm, 130 Å) during an elution window of 28 min, leading to a sufficient number of both data points across the chromatographic peaks, as well as MS2 spectra. Analysis was carried out in positive and negative ionization mode enabling the detection of a broader spectrum of lipids and to support the structural characterization of lipids. Optimal sample preparation of biological samples was achieved by liquid-liquid extraction using MeOH/MTBE resulting in an excellent extraction recovery > 85% with an intra-day and inter-day variability < 15%. The optimized method was applied on the investigation of changes in the phospholipid pattern in plasma from human subjects supplemented with n3-PUFA (20:5 and 22:6). The strongest increase was observed for lipids bearing 20:5, while 22:4 bearing lipids were lowered. Specifically, LPC 20:5_0:0 and PC 16:0_20:5 were found to be strongest elevated, while PE 18:0_22:4 and PC 18:2_18:2 were decreased by n3-PUFA supplementation. These results were confirmed by targeted LC-MS/MS using commercially available phospholipids as standards.

Do meals contain a relevant amount of oxylipins? LC-MS-based analysis of oxidized fatty acids in food.

Several oxylipins are potent lipid mediators and are discussed to be absorbed after oral intake. However, information about their concentrations in oils and processed foods are scarce. Here, we analyzed the concentrations of mono-, di- and multihydroxy- as well as epoxy-PUFA in virgin and refined oils as well as in different foods/meals. Oil refining causes hydrolysis of epoxy-PUFA and thus high dihydroxy-PUFA concentrations (e.g. 15,16-DiHODE 290 µg/g in refined vs. 15 µg/g in virgin rapeseed oil), making the epoxy-to-diol ratio a potential marker for refined oils. Low oxylipin levels were found in foods with high amounts of saturated fatty acids such as Hamburger patties (around 30 µg/g). High concentrations (up to 1200 µg/g, 80 mg per serving) and high oxylipin/precursor-PUFA ratios were found in fried falafel and processed foods such as vegetarian sausage/fish fingers. Our study provides first insights in the oxylipin concentrations of our daily food, indicating a relevant intake.

Beyond Autoxidation and Lipoxygenases: Fatty Acid Oxidation Products in Plant Oils.

For decades, research on oxidation of linoleic acid (LA, C18:2 n6) and α-linolenic acid (ALA, C18:3 n3) in plant oils has focused on autoxidatively formed and lipoxygenase-derived 9-hydro(pero)xy- and 13-hydro(pero)xy-LA and -ALA. Here, using a non-targeted approach, we show that other hydroxy fatty acids are more abundant in plant oils. Liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry analyses unveiled highly abundant peaks in flaxseed and rapeseed oils. Using authentic reference standards, seven of the peaks were identified as 9-, 10-, 12-, 13-, and 15-HODE as well as 9- and 13-HOTrE. Additionally, six peaks were characterized based on the retention time, the exact mass of the [M-H]- ion, and its fragment ions as 16-OH-C18:3, 18-OH-C18:3, three isomers of 12-OH-C18:2, and one of 15-OH-C18:2. 16-OH-C18:3 and 18-OH-C18:3 were tentatively identified as 16-OH-ALA and 18-OH-ALA, respectively, based on autoxidation and terminal hydroxylation of ALA using CYP4F2. Investigation of formation pathways suggests that fatty acid desaturase 3 is involved in the formation of the 12-OH-C18:2 isomers, 15-HODE, and its isomer. The dominantly occurring 12-OH-C18:2 isomer was identified as 12R,S-OH-9Z,15Z-octadecadienoic acid (densipolic acid) based on a synthetic standard. The characterized oxylipins occurred in cold-pressed flaxseed and rapeseed oils at concentrations of up to 0.1 g/100 g and thus about sixfold higher than the well-known 9-hydro(pero)xy- and 13-hydro(pero)xy-LA and -ALA. Concentrations in sunflower oil were lower but increased when oil was pressed from preheated seeds. Overall, this study provides fundamental new information about the occurrence of oxidized fatty acids in plant oils, having the potential to characterize their quality and authenticity.

Integrated analysis of whole blood oxylipin and cytokine responses after bacterial, viral, and T cell stimulation reveals new immune networks.

Oxylipins are major immunomodulating mediators, yet studies of inflammation focus mainly on cytokines. Here, using a standardized whole-blood stimulation system, we characterized the oxylipin-driven inflammatory responses to various stimuli and their relationships with cytokine responses. We performed a pilot study in 25 healthy individuals using 6 different stimuli: 2 bacterial stimuli (LPS and live BCG), 2 viral stimuli (vaccine-grade poly I:C and live H1N1 attenuated influenza), an enterotoxin superantigen and a Null control. All stimuli induced a strong production of oxylipins but most importantly, bacterial, viral, and T cell immune responses show distinct oxylipin signatures. Integration of the oxylipin and cytokine responses for each condition revealed new immune networks improving our understanding of inflammation regulation. Finally, the oxylipin responses and oxylipin-cytokine networks were compared in patients with active tuberculosis or with latent infection. This revealed different responses to BCG but not LPS stimulation highlighting new regulatory pathways for further investigations.

Prevention of colitis-induced liver oxidative stress and inflammation in a transgenic mouse model with increased omega-3 polyunsaturated fatty acids.

Inflammatory bowel disease (IBD) is an immune-mediated gut dysfunction, which might also be associated with an inflammatory phenotype in the liver. It is known that the nutritional intake of omega-3 polyunsaturated fatty acids (n-3 PUFA) is inversely correlated to the severity and occurrence of IBD. In order to investigate whether n-3 PUFA can also reduce liver inflammation and oxidative liver damage due to colon inflammation, we explored the dextran sulfate sodium (DSS)-induced colitis model in wild-type and fat-1 mice with endogenously increased n-3 PUFA tissue content. Besides confirming previous data of alleviated DSS-induced colitis in the fat-1 mouse model, the increase of n-3 PUFA also resulted in a significant reduction of liver inflammation and oxidative damage in colitis-affected fat-1 mice as compared to wild-type littermates. This was accompanied by a remarkable increase of established inflammation-dampening n-3 PUFA oxylipins, namely docosahexaenoic acid-derived 19,20-epoxydocosapentaenoic acid and eicosapentaenoic acid-derived 15-hydroxyeicosapentaenoic acid and 17,18-epoxyeicosatetraenoic acid. Taken together, these observations demonstrate a strong inverse correlation between the anti-inflammatory lipidome derived from n-3 PUFA and the colitis-triggered inflammatory changes in the liver by reducing oxidative liver stress.

Trans-Hydroxy, Trans-Epoxy, and Erythro-dihydroxy Fatty Acids Increase during Deep-Frying.

Deep-frying of food is a common cooking technique causing thermal oxidation of fatty acids (FA). Here, we investigated for the first time the formation of hydroxy-, epoxy- and dihydroxy-FA derived from oleic, linoleic (LA), and α-linolenic acid (ALA) during frying. Potato chips were fried in high-oleic sunflower oil for 4 × 5 cycles on 2 days, and the oil was comprehensively analyzed by liquid chromatography-tandem mass spectrometry. During frying, the E,Z-9- and E,Z-13-hydroperoxy-LA and -ALA concentrations decrease while their corresponding hydroxy-FA remain constant. The concentrations of both E,E-9-/13-hydroperoxy-LA and E,E-9-/13-hydroxy-LA increase with the frying cycles, which is also found for the concentration of trans-epoxy-FA. The increase in trans-epoxy-FA is more pronounced than that of the corresponding cis-epoxy-FA, exceeding their concentrations on the second day of frying. This selective change in the cis-/trans-epoxy-FA ratio is also observed for their hydrolysis products: concentrations of erythro-dihydroxy-FA, derived from trans-epoxy-FA, increase during frying stronger than threo-dihydroxy-FA derived from cis-epoxy-FA. Based on these data, we suggest that the ratio of E,E-/E,Z-hydroxy-FA, in combination with the cis-/trans-epoxy-FA ratio, as well as the threo-/erythro-dihydroxy-FA ratio are promising new parameters to evaluate the heating of edible oils and to characterize the status of frying oils.

Formation of lipoxins and resolvins in human leukocytes.

Specialized pro-resolving lipid mediators (SPMs) such as lipoxins or resolvins are formed by the consecutive action of 5-lipoxygenase (5-LO, ALOX5) and different types of arachidonic acid 12- or 15-lipoxygenases using arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid as substrate. Lipoxins are trihydroxylated oxylipins which are formed from arachidonic and eicosapentaenoic acid. The latter can also be converted to di- and trihydroxylated resolvins of the E series, whereas docosahexaenoic acid is the substrate for the formation of di- and trihydroxylated resolvins of the D series. Here, we summarize the formation of lipoxins and resolvins in leukocytes. From the data published so far, it becomes evident that FLAP is required for the biosynthesis of most of the lipoxins and resolvins. Even in the presence of FLAP, formation of the trihydroxylated SPMs (lipoxins, RvD1-RvD4, RvE1) in leukocytes is very low or undetectable which is obviously due to the extremely low epoxide formation by 5-LO from oxylipins such as 15-H(p)ETE, 18-H(p)EPE or 17-H(p)DHA. As a result, only the dihydroxylated oxylipins (5 S,15S-diHETE, 5 S,15S-diHEPE) and resolvins (RvD5, RvE2, RvE4) can be consistently detected using leukocytes as SPM source. However, the reported levels of these dihydroxylated lipid mediators are still much lower than those of the typical pro-inflammatory mediators including the monohydroxylated fatty acid derivatives (e.g. 5-HETE), leukotrienes or cyclooxygenase-derived prostaglandins. Since 5-LO expression is mainly restricted to leukocytes these cells are considered as the main source of SPMs. The low formation of trihydroxylated SPMs in leukocytes, the fact that they are hardly detected in biological samples as well as the lack of functional signaling by their receptors make it highly questionable that trihydroxylated SPMs play a role as endogenous mediators in the resolution of inflammation.

Sorafenib increases cytochrome P450 lipid metabolites in patient with hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer death, and medical treatment options are limited. The multikinase inhibitor sorafenib was the first approved drug widely used for systemic therapy in advanced HCC. Sorafenib might affect polyunsaturated fatty acids (PUFA)-derived epoxygenated metabolite levels, as it is also a potent inhibitor of the soluble epoxide hydrolase (sEH), which catalyzes the conversion of cytochrome-P450 (CYP)-derived epoxide metabolites derived from PUFA, such as omega-6 arachidonic acid (AA) and omega-3 docosahexaenoic acid (DHA), into their corresponding dihydroxy metabolites. Experimental studies with AA-derived epoxyeicosatrienoic acids (EETs) have shown that they can promote tumor growth and metastasis, while DHA-derived 19,20-epoxydocosapentaenoic acid (19,20-EDP) was shown to have anti-tumor activity in mice. In this study, we found a significant increase in EET levels in 43 HCC patients treated with sorafenib and a trend towards increased levels of DHA-derived 19,20-EDP. We demonstrate that the effect of sorafenib on CYP- metabolites led to an increase of 19,20-EDP and its dihydroxy metabolite, whereas DHA plasma levels decreased under sorafenib treatment. These data indicate that specific supplementation with DHA could be used to increase levels of the epoxy compound 19,20-EDP with potential anti-tumor activity in HCC patients receiving sorafenib therapy.

Adipocyte autophagy limits gut inflammation by controlling oxylipin and IL-10.

Lipids play a major role in inflammatory diseases by altering inflammatory cell functions, either through their function as energy substrates or as lipid mediators such as oxylipins. Autophagy, a lysosomal degradation pathway that limits inflammation, is known to impact on lipid availability, however, whether this controls inflammation remains unexplored. We found that upon intestinal inflammation visceral adipocytes upregulate autophagy and that adipocyte-specific loss of the autophagy gene Atg7 exacerbates inflammation. While autophagy decreased lipolytic release of free fatty acids, loss of the major lipolytic enzyme Pnpla2/Atgl in adipocytes did not alter intestinal inflammation, ruling out free fatty acids as anti-inflammatory energy substrates. Instead, Atg7-deficient adipose tissues exhibited an oxylipin imbalance, driven through an NRF2-mediated upregulation of Ephx1. This shift reduced secretion of IL-10 from adipose tissues, which was dependent on the cytochrome P450-EPHX pathway, and lowered circulating levels of IL-10 to exacerbate intestinal inflammation. These results suggest an underappreciated fat-gut crosstalk through an autophagy-dependent regulation of anti-inflammatory oxylipins via the cytochrome P450-EPHX pathway, indicating a protective effect of adipose tissues for distant inflammation.

Development of a quantitative proteomics approach for cyclooxygenases and lipoxygenases in parallel to quantitative oxylipin analysis allowing the comprehensive investigation of the arachidonic acid cascade.

Oxylipins derived from the cyclooxygenase (COX) and lipoxygenase (LOX) pathways of the arachidonic acid (ARA) cascade are essential for the regulation of the inflammatory response and many other physiological functions. Comprehensive analytical methods comprised of oxylipin and protein abundance analysis are required to fully understand mechanisms leading to changes within these pathways. Here, we describe the development of a quantitative multi-omics approach combining liquid chromatography tandem mass spectrometry-based targeted oxylipin metabolomics and proteomics. As the first targeted proteomics method to cover these pathways, it enables the quantitative analysis of all human COX (COX-1 and COX-2) and relevant LOX pathway enzymes (5-LOX, 12-LOX, 15-LOX, 15-LOX-2, and FLAP) in parallel to the analysis of 239 oxylipins with our targeted oxylipin metabolomics method from a single sample. The detailed comparison between MRM3 and classical MRM-based detection in proteomics showed increased selectivity for MRM3, while MRM performed better in terms of sensitivity (LLOQ, 16-122 pM vs. 75-840 pM for the same peptides), linear range (up to 1.5-7.4 µM vs. 4-368 nM), and multiplexing capacities. Thus, the MRM mode was more favorable for this pathway analysis. With this sensitive multi-omics approach, we comprehensively characterized oxylipin and protein patterns in the human monocytic cell line THP-1 and differently polarized primary macrophages. Finally, the quantification of changes in protein and oxylipin levels induced by lipopolysaccharide stimulation and pharmaceutical treatment demonstrates its usefulness to study molecular modes of action involved in the modulation of the ARA cascade.

GC-MS analysis of oxysterols and their formation in cultivated liver cells (HepG2).

Oxysterols play a key role in many (patho)physiological processes and they are potential biomarkers for oxidative stress in several diseases. Here we developed a rapid gas chromatographic-mass spectrometry-based method for the separation and quantification of 11 biologically relevant oxysterols bearing hydroxy, epoxy, and dihydroxy groups. Efficient chromatographic separation (resolution ≥ 1.9) was achieved using a medium polarity 35%-diphenyl/65%-dimethyl polysiloxane stationary phase material (30 m × 0.25 mm inner diameter and 0.25 µm film thickness). Based on thorough analysis of the fragmentation during electron ionization we developed a strategy to deduce structural information of the oxysterols. Optimized sample preparation includes (i) extraction with a mixture of n-hexane/iso-propanol, (ii) removal of cholesterol by solid phase extraction with unmodified silica, and (iii) trimethylsilylation. The method was successfully applied on the analysis of brain samples, showing consistent results with previous studies and a good intra- and interday precision of ≤20%. Finally, we used the method for the investigation of oxysterol formation during oxidative stress in HepG2 cells. Incubation with tert-butyl hydroperoxide led to a massive increase in free radical formed oxysterols (7-keto-chol > 7ß-OH-chol >> 7α-OH-chol), while 24 h incubation with the glutathione peroxidase 4 inhibitor RSL3 showed no increase in oxidative stress based on the oxysterol pattern. Overall, the new method described here enables the robust analysis of a biologically meaningful pattern of oxysterols with high sensitivity and precision allowing us to gain new insights in the biological formation and role of oxysterols.

Corrigendum: Sorafenib increases cytochrome P450 lipid metabolites in patient with hepatocellular carcinoma.

[This corrects the article DOI: 10.3389/fphar.2023.1124214.].