Oxylipin Concentrations Research Articles

Mass spectrometry unveils heat-induced changes in yolk oxylipins and key lipid molecules during home cooking

IntroductionOxylipins, as a widespread class of metabolic markers following oxidative stress, and several studies have reported dietary regulation of lipid metabolism. However, there is a lack of investigation of dietary oxylipins, especially cooking-induced changes in food lipid oxidation. ObjectivesInvestigated the effects of cooking methods and lipid profiles on polyunsaturated fatty acids derived oxylipins generation within egg yolks. MethodsThe lipid profile of egg yolk was determined by UPLC-QTOF-MS/MS, oxylipins were detected by HPLC-QTRAP-MS/MS, while the total fatty acid content was quantified by GC-FID. Random Forest (RF) and Partial Least Squares (PLS) regression models were employed to explore the association between oxidized lipids and key lipid species. ResultsHeating reduced egg yolk docosahexaenoic acid (DHA) content, and no consistent trends for arachidonic acid (AA), linoleic acid (LA), and linolenic acid (ALA). Yolk lipid composition affected triacylglycerol (TG), phosphatidylethanolamine (PE), and LA-monoepoxide contents after cooking. 9- and 13-HODE (hydroxyoctadecadienoic acid), 9,10,13-TriHOME (trihydroxyoctadecenoic acid), 9,10- and 12,13-EpOME (epoxyoctadecenoic acid), 9,10- and 12,13-DiHOME (dihydroxyoctadecenoic acid), 5-HETE (hydroxyeicosatetraenoic acid), and 4-HDHA (hydroxydocosahexaenoic acid) were the prevalent oxylipins with high concentrations, accounting for 1.08 %–29.58 % of the total content of 29 oxylipins. Steaming resulted in a 1.9-fold increase in oxylipin concentrations in yolks compared to raw yolks, and boiling with or without shells (poaching) resulted in a 1.30- to 1.76-fold increase in oxylipin concentrations. In contrast, pan-fried yolks exhibited the lowest and least variable levels of total oxylipins, while still retaining some epoxides, including epoxyeicosatrienoic acid (EET) and EpOME. Utilizing big data analysis, we mapped the oxylipin network in both ordinary and DHA-enriched egg yolks, revealing a strong correlation between cooking-induced oxylipin production and variations in 24 lipid species. ConclusionRevealed the potential mechanisms and key lipid molecules for heating-induced oxylipin production of yolk through lipidomics and big data analysis.

Oil polyunsaturated fatty acid composition but not tocopherol levels determine oxylipin formation in plant and algae oils during pan-frying

The oxidation of linoleic acid (LA) and α-linolenic acid (ALA) in plant oils results in primary oxidation products known as oxylipins. It is not known whether the mechanisms of oxidation are driven primarily by oil polyunsaturated fatty acid (PUFA) composition (i.e., more double bonds resulting in more rapid oxidation) or antioxidant (tocopherol) content. The present study addressed this unknown by measuring the effects of pan-frying on LA- and ALA-derived oxylipin concentrations in plant and algae oils with different baseline fatty acid and tocopherol concentrations. Oxylipins, free fatty acids and tocopherols were quantified in algae, olive, corn, canola, and soybean oils before and after 30 minutes of pan-frying. Fatty acid epoxides and hydroxides of LA or ALA were significantly elevated after pan-frying, whereas fatty acid diols were reduced. The level of change in oxylipins was lower in low-PUFA algae and extra virgin olive oils compared to soybean, canola and corn oils (high in PUFAs). Tocopherols decreased significantly after 30 minutes of pan-frying, particularly in oils with low PUFA composition. Minor changes were observed in free fatty acids. Our findings demonstrate that oil PUFA composition is a greater determinant of lipid oxidation than tocopherol content, and that PUFAs in high LA or ALA oils may spare tocopherol oxidation by acting as free radical scavengers.

Physiological changes associated with aging: Identification of novel biomarkers for frailty syndrome in women

This study explores the physiological changes associated with aging that lead to frailty syndrome, characterized by reduced vitality and degeneration across multiple bodily systems, increasing susceptibility to various pathologies. While established scales like the Fried Phenotype and Frailty Trait Scale (FTS) are commonly used for assessing frailty, incorporating biomarkers is crucial for accurate diagnosis and prognosis. Our research examines plasma oxylipin levels in frail elderly individuals to identify novel biomarkers. Diagnostic criteria for frailty included assessments using the Fried Phenotype and FTS-5, with blood samples collected from 71 elderly participants (50 women and 21 men) with mean ages of 73.6 ± 5.9 and 76.2 ± 6.2 years, respectively. Women exhibited elevated platelet counts (p-value 0.0035). The significant differences in oxylipin concentrations associated with the Fried Phenotype were particularly noteworthy, predominantly observed in women. Specifically, in women, decreased grip strength (<15 kg) and slow gait speed (<0.8 m/s) correlated with increased levels of thromboxane B2 (TxB2) and 7-HDoHE (p-values 0.0404, 0.0300, 0.0033, and 0.0033, respectively). Additionally, elevated 7-HDoHE levels correlated with a BMI exceeding 28 kg/m2 (p-value 0.0123) and Physical Activity Scale for the Elderly (PASE) scores surpassing 5 points (p-value 0.0134) in women. In summary, our findings emphasize that frail older individuals, particularly women, exhibit higher levels of TxB2 and 7-HDoHE compared to their non-frail counterparts, aligning with established frailty classification and scale parameters, suggesting their potential as indicative biomarkers.

Endometrial responsiveness to interferon-tau and its association with subsequent reproductive performance in dairy heifers

Our objectives were to evaluate the endometrial responsiveness of dairy heifers to an intrauterine infusion of recombinant bovine interferon-tau (rbIFN-τ) and to associate endometrial responses to rbIFN-τ with subsequent reproductive performance. In experiments 1 and 2, cyclic heifers were enrolled in a program for synchronization of the estrous cycle, and blood sampling and ultrasonography examinations were performed on d 0, 4, 7, 11, and 14 of the estrous cycle. In experiment 1, heifers were randomly assigned to receive an intrauterine infusion containing 2 µg of rbIFN-τ (rbIFN-τ = 19) or saline control (CTRL = 19) into the uterine horn ipsilateral to the corpus luteum (CL) on d 14 of the estrous cycle. Then, 6 hours after the infusion, the infused uterine horn was flushed for sampling of the uterine luminal fluid (ULF) for composition analysis, and the endometrium was biopsied for transcriptomics. In experiment 2, 100 heifers received an intrauterine infusion of rbIFN-τ, and the same procedures for uterine sample collection were performed as described in experiment 1. After the intrauterine test, heifers were enrolled in a breeding program and classified as highly fertile (HF; pregnant at first AI) or subfertile (SF; not pregnant at first AI). Statistical analyses were performed using regression models, which included the effects of treatment (experiment 1: CTRL vs. rbIFN-τ) or fertility group (experiment 2: HF vs. SF) and block of samples. Intrauterine infusion of rbIFN-τ increased the expression of classical interferon-stimulated genes in the endometrium (e.g., ISG15, MX1, OAS2, IRF9, and USP18), and an antiviral response was predicted to be the main downstream effect of the transcriptome changes. In addition, rbIFN-τ increased the abundance of cholesterol, glycerol, and the overall concentration of oxylipins in the ULF. Analysis of endometrial transcriptome between HF and SF heifers revealed important differences in the expression of genes associated with cell signaling, metabolism, attachment, and migration, with a large representation of genes encoding extracellular matrix proteins. In general, differentially expressed genes were expected to be downregulated by IFN-τ but seemed to fail to be downregulated in SF heifers, resulting in higher expression in SF compared with HF heifers. Subfertile heifers had lower concentrations of glycerol and an altered profile of oxylipins in the ULF, with a lower abundance of oxylipins derived from arachidonic acid and dihomo-γ-linolenic acid, and a greater abundance of oxylipins derived from linoleic acid. Measurements of ovarian function did not differ between groups and, therefore, did not influence the observed results in uterine biology. Overall, the endometrial responsiveness to IFN-τ is variable among individuals and associated with subsequent fertility of heifers, indicating that communication between conceptus and endometrium is critical for the uterine receptivity and survival of pregnancy.

Docosahexaenoic Acid Controls Pulmonary Macrophage Lipid Raft Size and Inflammation

BackgroundDocosahexaenoic acid (DHA) controls the biophysical organization of plasma membrane sphingolipid/cholesterol-enriched lipid rafts to exert anti-inflammatory effects, particularly in lymphocytes. However, the impact of DHA on the spatial arrangement of alveolar macrophage lipid rafts and inflammation is unknown. ObjectivesThe primary objective was to determine how DHA controls lipid raft organization and function of alveolar macrophages. As proof-of-concept, we also investigated DHA’s anti-inflammatory effects on select pulmonary inflammatory markers with a murine influenza model. MethodsMH-S cells, an alveolar macrophage line, were treated with 50 μM DHA or vehicle control and were used to study plasma membrane molecular organization with fluorescence-based methods. Biomimetic membranes and coarse grain molecular dynamic (MD) simulations were employed to investigate how DHA mechanistically controls lipid raft size. qRT-PCR, mass spectrometry, and ELISAs were used to quantify downstream inflammatory signaling transcripts, oxylipins, and cytokines, respectively. Lungs from DHA-fed influenza-infected mice were analyzed for specific inflammatory markers. ResultsDHA increased the size of lipid rafts while decreasing the molecular packing of the MH-S plasma membrane. Adding a DHA-containing phospholipid to a biomimetic lipid raft-containing membrane led to condensing, which was reversed with the removal of cholesterol. MD simulations revealed DHA nucleated lipid rafts by driving cholesterol and sphingomyelin into rafts. Downstream of the plasma membrane, DHA lowered the concentration of select inflammatory transcripts, oxylipins, and IL-6 secretion. DHA lowered pulmonary Il6 and Tnf-α mRNA expression and increased anti-inflammatory oxylipins of influenza-infected mice. ConclusionsThe data suggest a model in which the localization of DHA acyl chains to nonrafts is driving sphingomyelin and cholesterol molecules into larger lipid rafts, which may serve as a trigger to impede signaling and lower inflammation. These findings also identify alveolar macrophages as a target of DHA and underscore the anti-inflammatory properties of DHA for lung inflammation.

Profiling of Oxylipins as Markers of Oxidative Stress in Biological Samples.

Oxylipins are oxidized metabolites of polyunsaturated fatty acids (PUFAs). They represent a class of risk markers and/or therapeutic targets for diseases associated with inflammation, including cardiovascular disease and brain disorders. Because the biological activities of free PUFAs and oxylipins depend on their chemical structures and concentrations, monitoring PUFAs and oxylipin levels in biological systems is critical for understanding their roles in health and disease. Traditionally, accurate quantification of free PUFAs and oxylipins in biological samples was performed separately, as PUFAs are often 1000-fold more abundant than the derived oxidized fatty acids (oxylipins). This article describes a liquid chromatography multiple reaction monitoring tandem mass spectrometry method for the quantitative analysis of five free PUFAs and 88 oxylipins in various biological fluids, including plasma, platelet supernatants, and tissues. The same approach can also be used in conjunction with an alkaline hydrolysis step to quantify total oxylipins in fish oils. We observed that in some samples, linoleic acid levels in plasma and eicosapentaenoic acid and arachidonic acid levels in brain tissue were above the upper limit of quantification. To address this issue, we developed a data analysis method to obtain PUFA and oxylipin concentrations in these samples without additional sample preparation, thus significantly saving time and labor. © 2024 Wiley Periodicals LLC. Basic Protocol: Quantification of polyunsaturated fatty acids (PUFAs) and oxylipins using liquid chromatography multiple reaction monitoring tandem mass spectrometry Support Protocol 1: Preparation of internal standard mixed working solution Support Protocol 2: Preparation of standard mixed stock solution Support Protocol 3: Preparation of standard mixed working solution Alternate Protocol 1: Extraction and quantitation of free PUFAs and oxylipins from mouse brain tissue Alternate Protocol 2: Extraction and quantitation of total PUFAs and oxylipins from fish oil.

Predictors of upstream inflammation and oxidative stress pathways during early pregnancy

BackgroundInflammation and oxidative stress are critical to pregnancy, but most human study has focused on downstream, non-causal indicators. Oxylipins are lipid mediators of inflammation and oxidative stress that act through many biological pathways. Our aim was to characterize predictors of circulating oxylipin concentrations based on maternal characteristics. MethodsOur study was conducted among 901 singleton pregnancies in the LIFECODES Fetal Growth Study, a nested case-cohort with recruitment from 2007 to 2018. We measured a targeted panel of oxylipins in early pregnancy plasma and urine samples from several biosynthetic pathways, defined by the polyunsaturated fatty acid (PUFA) precursor and enzyme group. We evaluated levels across predictors, including characteristics of participants’ pregnancy, socioeconomic determinants, and obstetric and medical history. ResultsCurrent pregnancy and sociodemographic characteristics were the most important predictors of circulating oxylipins concentrations. Plasma oxylipins were lower and urinary oxylipins higher for participants with a later gestational age at sampling (13–23 weeks), higher prepregnancy BMI (obesity class I, II, or III), Black or Hispanic race and ethnicity, and lower socioeconomic status (younger age, lower education, and uninsured). For example, compared to those with normal or underweight prepregnancy BMI, participants with class III prepregnancy obesity had 45–46% lower plasma epoxy-eicosatrienoic acids, the anti-inflammatory oxylipins produced from arachidonic acid (AA) by cytochrome P450, and had 81% higher urinary 15-series F2-isoprostanes, an indicator of oxidative stress produced from non-enzymatic AA oxidation. Similarly, in urine, Black participants had 92% higher prostaglandin E2 metabolite, a pro-inflammatory oxylipin, and 41% higher 5-series F2-isoprostane, an oxidative stress indicator. ConclusionsIn this large pregnancy study, we found that circulating levels of oxylipins were different for participants of lower socioeconomic status or of a systematically marginalized racial and ethnic groups. Given associations differed along biosynthetic pathways, results provide insight into etiologic links between maternal predictors and inflammation and oxidative stress.

Impact of Reperfusion on Plasma Oxylipins in ST-Segment Elevation Myocardial Infarction.

ST-segment elevation myocardial infarction (STEMI) occurs as a result of acute occlusion of the coronary artery. Despite successful reperfusion using primary percutaneous coronary intervention (PPCI), a large percentage of myocardial cells die after reperfusion, which is recognized as ischemia/reperfusion injury (I/R). There are rapid changes in plasma lipidome during myocardial reperfusion injury. However, the impact of coronary artery reperfusion on plasma oxylipins is unknown. This study aimed to investigate alterations in the oxylipin profiles of STEMI patients during ischemia and at various reperfusion time points following PPCI. Blood samples were collected from patients presenting with STEMI prior to PPCI (Isch, n = 45) and subsequently 2 h following successful reperfusion by PPCI (R-2 h, n = 42), after 24 h (R-24 h, n = 44), after 48 h (R-48 h, n = 43), and then 30 days post PPCI (R-30 d, n = 29). As controls, blood samples were collected from age- and sex-matched patients with non-obstructive coronary artery disease after diagnostic coronary angiography. High-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) using deuterated standards was used to identify and quantify oxylipins. In patients presenting with STEMI prior to reperfusion (Isch group), the levels of docosahexaenoic acid (DHA)-derived oxylipins were significantly higher when compared with controls. Their levels were also significantly correlated with the peak levels of creatine kinase (CK) and troponin T(TnT) before reperfusion (CK: r = 0.33, p = 0.046, TnT: r = 0.50, p = 1.00 × 10-3). The total concentrations of oxylipins directly produced by 5-lipoxygenase (5-LOX) were also significantly elevated in the Isch group compared with controls. The ratio of epoxides (generated through epoxygenase) to diols (generated by soluble epoxide hydrolysis (sEH)) was significantly lower in the Isch group compared with the controls. Following reperfusion, there was an overall reduction in plasma oxylipins in STEMI patients starting at 24 h post PPCI until 30 days. Univariate receiver operating characteristic (ROC) curve analysis also showed that an elevated ratio of epoxides to diols during ischemia is a predictor of smaller infarct size in patients with STEMI. This study revealed a large alteration in plasma oxylipins in patients presenting with STEMI when compared with controls. Total oxylipin levels rapidly reduced post reperfusion with stable levels reached 24 h post reperfusion and maintained for up to 30 days post infarct. Given the shifts in plasma oxylipins following coronary artery reperfusion, further research is needed to delineate their clinical impact in STEMI patients.

Metabolic adaptations in severe obesity: Insights from circulating oxylipins before and after weight loss

BackgroundThe relationship between lipid mediators and severe obesity remains unclear. Our study investigates the impact of severe obesity on plasma concentrations of oxylipins and fatty acids and explores the consequences of weight loss. MethodsIn the clinical trial identifier NCT05554224 study, 116 patients with severe obesity and 63 overweight/obese healthy controls matched for age and sex (≈2:1) provided plasma. To assess the effect of surgically induced weight loss, we requested paired plasma samples from 44 patients undergoing laparoscopic sleeve gastrectomy one year after the procedure. Oxylipins were measured using ultra-high-pressure liquid chromatography coupled to a triple quadrupole mass spectrometer via semi-targeted lipidomics. Cytokines and markers of interorgan crosstalk were measured using enzyme-linked immunosorbent assays. ResultsWe observed significantly elevated levels of circulating fatty acids and oxylipins in patients with severe obesity compared to their metabolically healthier overweight/obese counterparts. Our findings indicated that sex and liver disease were not confounding factors, but we observed weak correlations in plasma with circulating adipokines, suggesting the influence of adipose tissue. Importantly, while weight loss restored the balance in circulating fatty acids, it did not fully normalize the oxylipin profile. Before surgery, oxylipins derived from lipoxygenase activity, such as 12-HETE, 11-HDoHE, 14-HDoHE, and 12-HEPE, were predominant. However, one year following laparoscopic sleeve gastrectomy, we observed a complex shift in the oxylipin profile, favoring species from the cyclooxygenase pathway, particularly proinflammatory prostanoids like TXB2, PGE2, PGD2, and 12-HHTrE. This transformation appears to be linked to a reduction in adiposity, underscoring the role of lipid turnover in the development of metabolic disorders associated with severe obesity. ConclusionsDespite the reduction in fatty acid levels associated with weight loss, the oxylipin profile shifts towards a predominance of more proinflammatory species. These observations underscore the significance of seeking mechanistic approaches to address severe obesity and emphasize the importance of closely monitoring the metabolic adaptations after weight loss.

Loss of 12-Lipoxygenase Improves the Post-Transfusion Function of Stored Platelets

Objective Platelets for transfusion are stored for 5-7 days. During storage, platelets undergo numerous detrimental functional changes. Previous studies have shown that HETE levels in the storage bag negatively correlate with platelet performance in vivo suggesting that the dysregulation of bioactive lipid mediators may contribute to the storage lesion. In the current study, we sought to understand how genetic deletion and pharmacologic inhibition of 12-lipoxygenase (12-LOX) affects platelets during storage and after transfusion. Approach and Results Platelets from12-LOX+/+ (wild-type [WT]) and 12-LOX-/- mice were stored for 24 and 48 hours and profiled using LC-MS/MS-MRM. Polyunsaturated fatty acids increased significantly in stored platelets from 12-LOX-/- mice, while oxylipin concentrations were significantly higher in WT platelets. Baseline αIIbβ 3 integrin activation was significantly lower before and after 24 hours of storage in 12-LOX-/- platelets than in WT platelets. Surprisingly, after transfusion to thrombocytopenic mice, we observed significantly more baseline αIIbβ3 integrin activation in 12-LOX-/- platelets than in WT platelets. Stored platelets from 12-LOX-/- mice occluded vessels significantly faster than stored WT platelets in thrombocytopenic recipients challenged with a FeCl 3-induced carotid artery injury. In hemostasis models, significantly more stored 12-LOX-/- than WT platelets accumulated at the site of venous injury leading to reduced blood loss. Inhibition of cyclooxygenase-1 (COX-1) with acetylsalicylic acid abrogated both increased integrin activation and thromboxane generation in stored 12-LOX-/- platelets, highlighting a critical role of this pathway for improved post-transfusion function. Consistent with our mouse studies, human platelets stored with the 12-LOX inhibitor, VLX-1005, showed increased integrin activation compared to vehicle-treated platelets upon transfusion in NOD/SCID mice. Conclusion Deleting 12-LOX improves the post-transfusion function of stored murine and human platelets by increasing thromboxane generation through a COX-1-dependent mechanism. Future studies should determine the feasibility and safety of 12-LOX-inhibited platelets transfused to humans.

Identification of omega-3 oxylipins in human milk-derived extracellular vesicles with pro-resolutive actions in gastrointestinal inflammation.

Premature infants (PIs) are at risk of suffering necrotizing enterocolitis (NEC), and infants consuming human milk (HM) show a lower incidence than infants receiving formula. The composition of HM has been studied in depth, but the lipid content of HM-derived small extracellular vesicles (HM sEVs) remains unexplored. Identifying these molecules and their biological effects has potential for the treatment of intestinal disorders in PIs and could contribute to the development of HM-based fortified formulas. We isolated HM sEVs from HM samples and analyzed their oxylipin content using liquid chromatography coupled to mass spectrometry, which revealed the presence of anti-inflammatory oxylipins. We then examined the efficacy of a mixture of these oxylipins in combating inflammation and fibrosis, in vitro and in a murine model of inflammatory bowel disease (IBD). HM-related sEVs contained higher concentrations of oxylipins derived from docosahexaenoic acid, an omega-3 fatty acid. Three anti-inflammatory oxylipins, 14-HDHA, 17-HDHA, and 19,20-DiHDPA (ω3 OXLP), demonstrated similar efficacy to HM sEVs in preventing cell injury, inducing re-epithelialization, mitigating fibrosis, and modulating immune responses. Both ω3 OXLP and HM sEVs effectively reduced inflammation in IBD-model mice, preventing colon shortening, infiltration of inflammatory cells and tissue fibrosis. Incorporating this unique cocktail of oxylipins into fortified milk formulas might reduce the risk of NEC in PIs and also provide immunological and neurodevelopmental support.

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.

Determination of oxylipins and their precursors in breast milk by solid phase extraction-ultra high performance liquid chromatography-triple quadrupole tandem mass spectrometry

Oxylipins and their precursors (long-chain polyunsaturated fatty acids, LCPUFAs) are key intercellular signaling molecules influencing the inflammatory response. Each oxylipin has pro- and/or anti-inflammatory effects, and the relative abundance of different oxylipins can alter the inflammatory balance, making it important to clarify the oxylipin profile of breast milk for optimal infant health. The extraction, identification, and simultaneous quantification of oxylipins in breast milk are challenging due to the structural similarity, limited stability, and the low endogenous concentration of oxylipins and the complex matrix of breast milk. This study aimed to develop a solid phase extraction-ultra high performance liquid chromatography-triple quadrupole tandem mass spectrometry (SPE-UPLC-MS/MS) method for the comprehensive and specific quantification of oxylipins and their precursors in breast milk. The LC conditions (including column, mobile phase, and gradient conditions) and SPE procedure (including SPE cartridges, elution solvent, and elution volume) were optimized to achieve accurate quantification and better analyte recovery. A single 18-minute chromatographic run allows for the quantification of 20 oxylipins and 5 PUFAs. The results showed good linearity (R2 > 0.99) over the concentration range of 2 to 100 ng/mL, with the instrument detection limits ranging from 0.01 to 0.90 ng/mL for oxylipins and 0.02 to 0.59 ng/mL for PUFAs. The method is rapid, sensitive, and reproducible (RSD ≤ 10%) and is suitable for the quantitative analysis of oxylipins and their precursors in infant formula samples.

Loss of 12-Lipoxygenase Improves the Post-Transfusion Function of Stored Platelets.

Platelets for transfusion are stored for 5 to 7 days. Previous studies have shown that HETE levels in the storage bag negatively correlate with platelet performance in vivo, suggesting that the dysregulation of bioactive lipid mediators may contribute to the storage lesion. In the current study, we sought to understand how genetic deletion and pharmacological inhibition of 12-LOX (12-lipoxygenase) affects platelets during storage and after transfusion. Platelets from 12-LOX+/+ (wild-type [WT]) and 12-LOX-/- mice were stored for 24 and 48 hours and profiled using liquid chromatography-tandem mass spectrometry-multiple reaction monitoring or transfused into thrombocytopenic hIL4R (human interleukin 4 receptor)-transgenic mice. Platelet function was assessed by flow cytometry and in vivo thrombosis and hemostasis models. To test the role of the COX-1 (cyclooxygenase-1) pathway, donor mice were treated with acetylsalicylic acid. Human platelets were treated with the 12-LOX inhibitor, VLX-1005, or vehicle, stored, and transfused to NOD/SCID (nonobese diabetic/severe combined immunodeficiency) mice. Polyunsaturated fatty acids increased significantly in stored platelets from 12-LOX-/- mice, whereas oxylipin concentrations were significantly higher in WT platelets. After transfusion to thrombocytopenic mice, we observed significantly more baseline αIIbβ3 integrin activation in 12-LOX-/- platelets than in WT platelets. Stored platelets from 12-LOX-/- mice occluded vessels significantly faster than stored WT platelets. In hemostasis models, significantly more stored 12-LOX-/- than WT platelets accumulated at the site of venous injury leading to reduced blood loss. Inhibition of COX-1 abrogated both increased integrin activation and thromboxane generation in stored 12-LOX-/- platelets, highlighting the critical role of this pathway for improved post-transfusion function. Consistent with our mouse studies, human platelets stored with VLX-1005, showed increased integrin activation compared with vehicle-treated platelets after transfusion. Deleting 12-LOX improves the post-transfusion function of stored murine platelets by increasing thromboxane generation through COX-1-dependent arachidonic acid metabolism. Future studies should determine the feasibility and safety of 12-LOX-inhibited platelets transfused to humans.

637-P: Sex-Specific Associations between Fatty Acid–Derived Oxylipins and Executive Function in T2DM

T2DM increases the risk of cognitive decline, especially in women. Sex-specific associations between lipids and cognition have been reported, though underlying mechanisms remain unclear. Disruptions in the cytochrome P450-soluble epoxide hydrolase (CYP450-sEH) pathway have been linked to both T2DM and cognitive dysfunction. CYP450s convert long-chain polyunsaturated fatty acids into epoxides which are anti-inflammatory and help resolve inflammation, however sEH metabolizes these into inactive or cytotoxic diols, limiting their benefits. We hypothesized that oxylipin epoxides and diols of arachidonic acid (AA) and docosahexaenoic acid (DHA) would be associated with cognition in people with T2DM, particularly in women. Women and men with T2DM were recruited as part of the study. Twenty-two fasting serum free oxylipins were quantified by ultra-high performance liquid chromatography tandem mass spectrometry. Executive function was assessed using a composite z-score from the following tests: Stroop Colour-Word Interference Victoria Version, FAS Verbal Fluency, Digit Symbol Substitution, and Trails Making Test Part B. All analyses controlled for age, sex, BMI, HbA1c and years of education. Among 72 individuals with T2DM (mean age 61.8±9.2, mean HbA1c 7.4±0.1%, 53% women), demographic characteristics, oxylipin concentrations and cognitive performance did not differ between women and men. Some CYP450-derived epoxides of DHA and AA were associated with lower executive function scores in women, but with higher scores in men. Epoxide × sex interactions were found for 3 of 4 DHA epoxides (16(17) [F=20.2, p&amp;lt;0.001]; 13(14) [F=8.5, p=0.005]; and 10(11) [F=7.4, p=0.009]), and for the 14(15) AA epoxide [F=3.9, p&amp;lt;0.05]). Circulating concentrations of the corresponding diols were not associated with executive function. These associations between epoxides and executive function suggest a sex difference in the relevance of the CYP450 pathway to cognitive performance in T2DM. Disclosure S.Ryoo: None. B.R.Shah: None. J.Gilbert: Speaker's Bureau; Abbott, AstraZeneca, Amgen Canada, Bayer Inc., Boehringer Ingelheim (Canada) Ltd., Sanofi, Dexcom, Inc., Eli Lilly and Company, HLS Therapeutics Inc., Novo Nordisk Canada Inc. A.Assal: None. I.Halperin: Advisory Panel; Sanofi, Speaker's Bureau; 3Boehringer Ingelheim Canada Ltd./Ltée, Abbott Diabetes, Dexcom, Inc., Novo Nordisk. A.Taha: None. W.Swardfager: None. N.Z.Anita: None. C.Major-orfao: None. W.Lin: None. S.Noor: None. F.Kwan: None. K.L.Lanctôt: Consultant; Exciva, GW Pharmaceuticals, ICG Farma, Merck Sharp &amp; Dohme Corp., Novo Nordisk, Sumitomo Pharma Co. Ltd., Other Relationship; BioXcel, Cerevel Therapeutics, H Lundbeck A/S. N.Herrmann: None. P.Oh: Advisory Panel; Novartis Canada. Funding Canadian Institutes of Health Research; Banting &amp; Best Diabetes Centre; National Institute of Diabetes and Digestive and Kidney Diseases

Oxylipin secretion by human CD3+ T lymphocytes in vitro is modified by the exogenous essential fatty acid ratio and life stage.

Immune function changes across the life stages; for example, senior adults exhibit a tendency towards a weaker cell-mediated immune response and a stronger inflammatory response than younger adults. This might be partly mediated by changes in oxylipin synthesis across the life course. Oxylipins are oxidation products of polyunsaturated fatty acids (PUFAs) that modulate immune function and inflammation. A number of PUFAs are precursors to oxylipins, including the essential fatty acids (EFAs) linoleic acid (LA) and α-linolenic acid (ALA). LA and ALA are also substrates for synthesis of longer chain PUFAs. Studies with stable isotopes have shown that the relative amounts of LA and ALA can influence their partitioning by T lymphocytes between conversion to longer chain PUFAs and to oxylipins. It is not known whether the relative availability of EFA substrates influences the overall pattern of oxylipin secretion by human T cells or if this changes across the life stages. To address this, the oxylipin profile was determined in supernatants from resting and mitogen activated human CD3+ T cell cultures incubated in medium containing an EFA ratio of either 5:1 or 8:1 (LA : ALA). Furthermore, oxylipin profiles in supernatants of T cells from three life stages, namely fetal (derived from umbilical cord blood), adults and seniors, treated with the 5:1 EFA ratio were determined. The extracellular oxylipin profiles were affected more by the EFA ratio than mitogen stimulation such that n-3 PUFA-derived oxylipin concentrations were higher with the 5:1 EFA ratio than the 8:1 ratio, possibly due to PUFA precursor competition for lipoxygenases. 47 oxylipin species were measured in all cell culture supernatants. Extracellular oxylipin concentrations were generally higher for fetal T cells than for T cells from adult and senior donors, although the composition of oxylipins was similar across the life stages. The contribution of oxylipins towards an immunological phenotype might be due to the capacity of T cells to synthesize oxylipins rather than the nature of the oxylipins produced.

Harmonization of OF LC‐MS oxylipin analysis to investigate lipid mediator formation and signaling in immune cells

The oxidation products of polyunsaturated fatty acids (PUFA) ‐ oxylipins ‐ are formed via enzymatic conversion by cyclooxygenases, lipoxygenases (LOX) and cytochrome P450 monooxygenases or by autoxidation. Together with multiple PUFA substrates this leads to a large variety of oxylipins, whose physiological functions are ‐ besides those of well‐studied prostaglandins and leukotrienes ‐ little understood. The interplay of several PUFA‐oxidizing enzymes, as in the case of multihydroxylated oxylipins, additionally complicates the investigation of their biosynthesis. Due to regulatory crosstalk and complex interactions of different oxylipin classes during physiological processes, such as inflammation, a study of the whole oxylipin profile rather than individual compounds is necessary. Therefore, in order to understand the functions of these lipid mediators potent and reliable biochemical methods and (molecular) biological models are required, in combination with sensitive and sound preanalytical, analytical and post‐analytical procedures for their investigation.The first part of this thesis investigated the relevance of harmonizing such analytical methods for the quantitative determination of oxylipins in biological samples. Based on the outcome of an international laboratory comparison, comparable and reproducible results were obtained across independent laboratories when these harmonized methods were used for sample preparation and analysis by means of liquid chromatography coupled with tandem mass spectrometry. The examination of the effect of different storage times and temperatures as a parameter of pre‐analytics demonstrated that oxylipin levels in plasma samples are robust to long‐term storage and, furthermore, to minor variations during plasma generation. As accurate quantification of oxylipin concentrations rises and falls with the quality of analytical standards, a strategy for the verification of oxylipin standard concentrations with mass‐ spectrometry and UV spectroscopy based approaches using certified standard compounds was presented.In the second part of the thesis the established methods were applied to study the biosynthesis of oxylipins in immune cells with a special attention to the involved LOX. Stimulation of primary human M2‐like macrophages with the liver X receptor (LXR) agonist T09 led to a massive induction of ALOX15 gene expression and thus, 15‐LOX abundance and activity. Cholesterol derivatives were identified as potent endogenous ligands of LXR yielding increased 15‐LOX abundance and activity. The examination of 5‐LOX induced synthesis of multihydroxylated metabolites through supplementation of neutrophils with 15‐HETE, 18‐ HEPE and 17‐HDHA primary leads to formation of double oxygenated oxylipins. In intact cells 5‐LOX preferred DHA‐derived 17‐HDHA as substrate, whereas upon cell integrity destruction the formation of 15‐HETE derived multihydroxylated oxylipins increased.With the combination of harmonized protocols for sampling and analysis this thesis sets the basis for the reliable quantification of oxylipins. The use of the optimized methodologies allowed to further characterize regulatory pathways of the ARA cascade in human immune cells, contributing to a more thorough understanding of inflammation regulation.

Concentration of milk oxylipins after heat and homogenization treatments

Heat treatment and homogenization of milk are common processing steps intended to reduce microbial load for safe human consumption, and to avoid creaming, respectively. Although the effects of combined pasteurization and homogenization on free fatty acids (FFA) and lipid oxidation markers such as hydroperoxides, and thiobarbituric acid reactive species (TBARS) have been well characterized, their effects on primary oxidized fatty acids known as oxylipins have not yet been determined. This study aimed to determine the effects of two heat treatments: milk pasteurization [high-temperature short time, 72°C for 15 s (HTST)] and sterilization [ultra-high temperature, 135°C for 3 s (UHT)] with or without homogenization (10, 17 or 24 MPa) on FFA (%), primary (hydroperoxides and oxylipins) and secondary oxidation compounds (TBARS). Heat treatments alone significantly reduced most oxylipins compared with raw milk, but did not alter % FFA, hydroperoxide, and TBARS levels. The combination of UHT and homogenization at 24 MPa increased % FFA, hydroperoxide value, and some oxylipins, compared to raw milk and other treatments. Overall, the combination of heat treatment and homogenization significantly increased oxylipin formation compared with heat treatment alone.

Twelve Weeks of High-Intensity Interval Training Alters Adipose Tissue Gene Expression but Not Oxylipin Levels in People with Non-Alcoholic Fatty Liver Disease.

Lifestyle modifications, including increased physical activity and exercise, are recommended for non-alcoholic fatty liver disease (NAFLD). Inflamed adipose tissue (AT) contributes to the progression and development of NAFLD and oxylipins such as hydroxyeicosatetraenoic acids (HETE), hydroxydocosahexanenoic acids (HDHA), prostaglandins (PEG2), and isoprostanoids (IsoP), which all may play a role in AT homeostasis and inflammation. To investigate the role of exercise without weight loss on AT and plasma oxylipin concentrations in NAFLD subjects, we conducted a 12-week randomized controlled exercise intervention. Plasma samples from 39 subjects and abdominal subcutaneous AT biopsy samples from 19 subjects were collected both at the beginning and the end of the exercise intervention. In the AT of women, a significant reduction of gene expression of hemoglobin subunits (HBB, HBA1, HBA2) was observed within the intervention group during the 12-week intervention. Their expression levels were negatively associated with VO2max and maxW. In addition, pathways involved in adipocyte morphology alterations significantly increased, whereas pathways in fat metabolism, branched-chain amino acids degradation, and oxidative phosphorylation were suppressed in the intervention group (p < 0.05). Compared to the control group, in the intervention group, the ribosome pathway was activated, but lysosome, oxidative phosphorylation, and pathways of AT modification were suppressed (p < 0.05). Most of the oxylipins (HETE, HDHA, PEG2, and IsoP) in plasma did not change during the intervention compared to the control group. 15-F2t-IsoP significantly increased in the intervention group compared to the control group (p = 0.014). However, this oxylipin could not be detected in all samples. Exercise intervention without weight loss may influence the AT morphology and fat metabolism at the gene expression level in female NAFLD subjects.

Exogenous Application of Salicylic Acid Modulates Oxidative Stress during the Seed Development of Rice (Oryza sativa L.) Grain

The present study aimed to discover the effects of exogenously foliar-applied salicylic acid (SA) in concentrations of 0, 1 and 15 mM (applied 7 and 21 days after heading) on oxidative stress. The effects were monitored through the concentrations of phytoprostanes (PhytoPs) and phytofurans (PhytoFs) in immature and mature grains of three genotypes of rice (‘R52’, ‘R45’ and ‘Yerua’), and their influence on grains per panicle and chalkiness. Chromatographic separation of PhytoPs and PhytoFs was performed using a UHPLC coupled to triple quadrupole-MS/MS (Agilent Technologies, Germany). The concentrations of oxylipins showed differences in both harvest times (immature and mature) for each genotype. The advanced lines, ‘R52’ and ‘R45’, showed concentrations that were 24.0 and 79.0% lower than those of the immature grains, respectively. The PhytoFs concentration in “R45” was 46.0% lower in the mature grains. In unripe grains, SA reduced a single oxylipin of all those analyzed, while in mature grains, a significant decrease in six of the ten monitored biomarkers was observed. The SA produced an increase in grains per panicle, and a decrease in chalkiness. Therefore, salicylic acid-mediated antioxidant regulatory capacities due to oxylipin down-regulation could favor grain filling and, hence, rice production.