Phosphatidylethanolamine Species Research Articles

Multiple activities of sphingomyelin synthase 2 generate saturated fatty acid- and/or monounsaturated fatty acid-containing diacylglycerol

Phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) (EC 3.1.4.3) and phosphatidylethanolamine (PE)-specific PLC (PE-PLC) (EC 3.1.4.62), which generate diacylglycerol (DG) and are tricyclodecan-9-yl-xanthogenate (D609)-sensitive, were detected in detergent-insoluble fractions of mammalian tissues approximately 70 and 35 years ago, respectively. However, the genes and proteins involved in PC-PLC and PE-PLC activities remain unknown. In a recent study, we observed that mammalian sphingomyelin synthase (SMS) 1 and SMS-related protein (SMSr) display PC-PLC and PE-PLC activities in vitro. In the present study, we showed that human SMS2, which is located in detergent-insoluble fractions of the plasma membrane, also possesses PC-PLC activity (approximately 41% of SMS activity), PE-PLC activity (approximately 4%), ceramide phosphoethanolamine synthase (CPES) activity (approximately 46%), and SMS activity in the presence of phospholipid-detergent mixed micelles. Moreover, purified SMS2 reconstituted in detergent-free proteoliposomes (near-native environments) showed PC-PLC, PE-PLC, and CPES activities. Notably, in the presence of approximately 2 mol% ceramide and 4 mol% PC (1:2 ratio), PC-PLC activity was almost equal to SMS activity. SMS2 as PC/PE-PLC showed substrate selectivity for saturated fatty acid- and/or monounsaturated fatty acid-containing PC and PE species. The PC-PLC/SMS inhibitor D609 inhibited all enzyme activities (SMS, PC-PLC, PE-PLC, and CPES) of SMS2. Moreover, Zn2+ strongly inhibited all the enzymatic activities of SMS2. Interestingly, DG inhibited the SMS activity of SMS2 (feedback control). These results indicate that mammalian SMS2 has unique enzymatic properties and is a candidate for a long-sought mammalian PC/PE-PLC.

Phosphatidylethanolamines link ferroptosis and autophagy during appressorium formation of rice blast fungus.

A cell death pathway, ferroptosis, occurs in conidial cells and is critical for formation and function of the infection structure, the appressorium, in the rice blast fungus Magnaporthe oryzae. In this study, we identified an orthologous lysophosphatidic acid acyltransferase (Lpaat) acting at upstream of phosphatidylethanolamines (PEs) biosynthesis and which is required for such fungal ferroptosis and pathogenicity. Two PE species, DOPE and SLPE, that depend on Lpaat function for production were sufficient for induction of lipid peroxidation and the consequent ferroptosis, thus positively regulating fungal pathogenicity. On the other hand, both DOPE and SLPE positively regulated autophagy. Loss of the LPAAT gene led to a decrease in the lipidated form of the autophagy protein Atg8, which is probably responsible for the autophagy defect of the lpaatΔ mutant. GFP-Lpaat was mostly localized on the membrane of lipid droplets (LDs) that were stained by the fluorescent dye monodansylpentane (MDH), suggesting that LDs serve as a source of lipids for membrane PE biosynthesis and probably as a membrane source of autophagosome. Overall, our results reveal novel intracellular membrane-bound organelle dynamics based on Lpaat-mediated lipid metabolism, providing a temporal and spatial link of ferroptosis and autophagy.

Effects of short-term supplementation with DHA-enriched phosphatidylcholine and phosphatidylserine on lipid profiles in the brain and liver of n-3 PUFA-deficient mice in early life after weaning.

Lack of n-3 polyunsaturated fatty acids during the period of maternity drastically lowers the docosahexaenoic acid (DHA) level in the brain of offspring and studies have demonstrated that different molecular forms of DHA are beneficial to brain development. The aim of this study was to investigate the effect of short-term supplementation with DHA-enriched phosphatidylserine (PS) and phosphatidylcholine (PC) on DHA levels in the liver and brain of congenital n-3-deficient mice. Dietary supplementation with DHA significantly changed the fatty acid composition of various phospholipid molecules in the cerebral cortex and liver while DHA-enriched phospholipid was more effective than DHA triglyceride (TG) in increasing brain and liver DHA. Both DHA-PS and DHA-PC could effectively increase the DHA levels, but DHA in the PS form was superior to PC in the contribution of DHA content in the brain ether-linked PC (ePC) and liver lyso-phosphatidylcholine molecular species. DHA-PC showed more significant effects on the increase of DHA in liver TG, PC, ePC, phosphatidylethanolamine (PE) and PE plasmalogen (pPE) molecular species and decreasing the arachidonic acid level in liver PC plasmalogen, ePC, PE and pPE molecular species compared with DHA-PS. The effect of dietary interventions with different molecular forms of DHA for brain and liver lipid profiles is different, which may provide theoretical guidance for dietary supplementation of DHA for people. © 2024 Society of Chemical Industry.

Circulating perturbation of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) is associated to cardiac remodeling and NLRP3 inflammasome in cardiovascular patients with insulin resistance risk

Lipidome perturbation occurring during meta-inflammation is associated to left ventricle (LV) remodeling though the activation of the NLRP3 inflammasome, a key regulator of chronic inflammation in obesity-related disorders. Little is known about phosphatidylcholine (PC) and phosphatidylethanolamine (PE) as DAMP-induced NLRP3 inflammasome. Our study is aimed to evaluate if a systemic reduction of PC/PE molar ratio can affect NLRP3 plasma levels in cardiovascular disease (CVD) patients with insulin resistance (IR) risk.Forty patients from IRCCS Policlinico San Donato were enrolled, and their blood samples were drawn before heart surgery. LV geometry measurements were evaluated by echocardiography and clinical data associated to IR risk were collected. PC and PE were quantified by ESI-MS/MS. Circulating NLRP3 was quantified by an ELISA assay.Our results have shown that CVD patients with IR risk presented systemic lipid impairment of PC and PE species and their ratio in plasma was inversely associated to NLRP3 levels. Interestingly, CVD patients with IR risk presented LV changes directly associated to increased levels of NLRP3 and a decrease in PC/PE ratio in plasma, highlighting the systemic effect of meta-inflammation in cardiac response. In summary, PC and PE can be considered bioactive mediators associated to both the NLRP3 and LV changes in CVD patients with IR risk.

Tafazzin deficiency causes substantial remodeling in the lipidome of a mouse model of Barth Syndrome cardiomyopathy.

Barth Syndrome (BTHS) is a rare X-linked disease, characterized clinically by cardiomyopathy, skeletal myopathy, neutropenia, and growth retardation. BTHS is caused by mutations in the phospholipid acyltransferase tafazzin (Gene: TAFAZZIN, TAZ). Tafazzin catalyzes the final step in the remodeling of cardiolipin (CL), a glycerophospholipid located in the inner mitochondrial membrane. As the phospholipid composition strongly determines membrane properties, correct biosynthesis of CL and other membrane lipids is essential for mitochondrial function. Mitochondria provide 95% of the energy demand in the heart, particularly due to their role in fatty acid oxidation. Alterations in lipid homeostasis in BTHS have an impact on mitochondrial membrane proteins and thereby contribute to cardiomyopathy. We analyzed a transgenic TAFAZZIN-knockdown (TAZ-KD) BTHS mouse model and determined the distribution of 193 individual lipid species in TAZ-KD and WT hearts at 10 and 50weeks of age, using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Our results revealed significant lipid composition differences between the TAZ-KD and WT groups, indicating genotype-dependent alterations in most analyzed lipid species. Significant changes in the myocardial lipidome were identified in both young animals without cardiomyopathy and older animals with heart failure. Notable alterations were found in phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysophosphatidylethanolamine (LPE), lysophosphatidylcholine (LPC) and plasmalogen species. PC species with 2-4 double bonds were significantly increased, while polyunsaturated PC species showed a significant decrease in TAZ-KD mice. Furthermore, Linoleic acid (LA, 18:2) containing PC and PE species, as well as arachidonic acid (AA, 20:4) containing PE 38:4 species are increased in TAZ-KD. We found higher levels of AA containing LPE and PE-based plasmalogens (PE P-). Furthermore, we are the first to show significant changes in sphingomyelin (SM) and ceramide (Cer) lipid species Very long-chained SM species are accumulating in TAZ-KD hearts, whereas long-chained Cer and several hexosyl ceramides (HexCer) species accumulate only in 50-week-old TAZ-KD hearts These findings offer potential avenues for the diagnosis and treatment of BTHS, presenting new possibilities for therapeutic approaches.

Spatial Neurolipidomics at the Single Amyloid-β Plaque Level in Postmortem Human Alzheimer's Disease Brain.

Lipid dysregulations have been critically implicated in Alzheimer's disease (AD) pathology. Chemical analysis of amyloid-β (Aβ) plaque pathology in transgenic AD mouse models has demonstrated alterations in the microenvironment in the direct proximity of Aβ plaque pathology. In mouse studies, differences in lipid patterns linked to structural polymorphism among Aβ pathology, such as diffuse, immature, and mature fibrillary aggregates, have also been reported. To date, no comprehensive analysis of neuronal lipid microenvironment changes in human AD tissue has been performed. Here, for the first time, we leverage matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) through a high-speed and spatial resolution commercial time-of-light instrument, as well as a high-mass-resolution in-house-developed orbitrap system to characterize the lipid microenvironment in postmortem human brain tissue from AD patients carrying Presenilin 1 mutations (PSEN1) that lead to familial forms of AD (fAD). Interrogation of the spatially resolved MSI data on a single Aβ plaque allowed us to verify nearly 40 sphingolipid and phospholipid species from diverse subclasses being enriched and depleted, in relation to the Aβ deposits. This included monosialo-gangliosides (GM), ceramide monohexosides (HexCer), ceramide-1-phosphates (CerP), ceramide phosphoethanolamine conjugates (PE-Cer), sulfatides (ST), as well as phosphatidylinositols (PI), phosphatidylethanolamines (PE), and phosphatidic acid (PA) species (including Lyso-forms). Indeed, many of the sphingolipid species overlap with the species previously seen in transgenic AD mouse models. Interestingly, in comparison to the animal studies, we observed an increased level of localization of PE and PI species containing arachidonic acid (AA). These findings are highly relevant, demonstrating for the first time Aβ plaque pathology-related alteration in the lipid microenvironment in humans. They provide a basis for the development of potential lipid biomarkers for AD characterization and insight into human-specific molecular pathway alterations.

Acute myocardial infarction preferentially alters low-abundant, long-chain unsaturated phospholipid and sphingolipid species in plasma high-density lipoprotein subpopulations

AimHigh-density lipoprotein (HDL) particles in ST-segment elevation myocardial infarction (STEMI) are deficient in their anti-atherogenic function. Molecular determinants of such deficiency remain obscure. MethodsFive major HDL subpopulations were isolated using density-gradient ultracentrifugation from STEMI patients (n = 12) and healthy age- and sex-matched controls (n = 12), and 160 species of phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, sphingomyelin and ceramide were quantified by LC-MS/MS. ResultsMultiple minor species of proinflammatory phosphatidic acid and lysophosphatidylcholine were enriched by 1.7–27.2-fold throughout the majority of HDL subpopulations in STEMI. In contrast, minor phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, sphingomyelin and ceramide species were typically depleted up to 3-fold in STEMI vs. control HDLs, while abundances of their major species did not differ between the groups. Intermediate-to-long-chain phosphatidylcholine, phosphatidylinositol and phosphatidylglycerol species were more affected by STEMI than their short-chain counterparts, resulting in positive correlations between their fold decrease and the carbon chain length. Additionally, fold decreases in the abundances of multiple lipid species were positively correlated with the double bond number in their carbon chains. Finally, abundances of several phospholipid and ceramide species were positively correlated with cholesterol efflux capacity and antioxidative activity of HDL subpopulations, both reduced in STEMI vs controls. KEGG pathway analysis tied these species to altered glycerophospholipid and linoleic acid metabolism. ConclusionsMinor unsaturated intermediate-to-long-chain phospholipid and sphingolipid species in HDL subpopulations are most affected by STEMI, reflecting alterations in glycerophospholipid and linoleic acid metabolism with the accumulation of proinflammatory lysolipids and maintenance of homeostasis of major phospholipid species.

The Role of G0S2 in Lipid Metabolism Regulation in Chronic Myeloid Leukemia

Introduction: Chronic myeloid leukemia (CML) can be effectively treated with tyrosine kinase inhibitors (TKIs) targeting BCR::ABL1, but resistance remains a clinical problem. TKIs do not target the quiescent CML leukemic stem cell (LSC), and many patients are treated for life at a high economic burden and sometimes with significant side effects. We recently published a tumor suppressor role for G0/G1 switch gene 2 (G0S2) in CML. Our data showed that loss of G0S2 expression in CML promoted disease progression and drug resistance by disrupting glycerophospholipid metabolism (Gonzalezet al. Clin Transl Med, 2022). Interestingly, G0S2 is an estrogen-response gene that demonstrated anti-estrogenic and pro-migratory effects in ER+ versus ER- breast cancer. Additionally, deletion of G0S2 prevented obesity and insulin resistance in mouse models. Since sex-based differences in lipid metabolism and obesity are well documented, we hypothesized that the tumor suppressor role of G0S2 in CML would differ based on sex. Methods: We used peripheral blood from wild-type and G0s2 knockout ( G0s2 -/-) mice (both male and female) who were fed with a normal versus a high-fat diet. Resulting lineage-negative (Lin-) cells were cultured in recombinant murine cytokines ± granulocyte-colony stimulating factor (mG-CSF, 25 ng/ml, 7-10 days) in in vitro differentiation assays. Flow cytometry analysis was performed to measure CD11b+ neutrophils. Additionally, we transduced Lin- cells from wild-type or G0s2 knockout mice with BCR::ABL1, and assessed their ability to form a leukemia-like disease process in male versus female recipient mice. We also performed G0S2 ectopic expression and knockdown experiments in the K562 CML cell line and assessed the resulting cells by liquid chromatography (LC)/mass spectrometry (MS)-based lipidomics and proteomics analyses. Immunoblots were performed to assess the resulting cells for changes in the autophagy markers, LC3A/B I-II. Results: For the experiments conducted with mouse peripheral blood, RT-qPCR data showed high G0S2 mRNA expression upon neutrophil differentiation in wild-type male mice (n=5), but not in the wild-type female mice (n=5). Flow cytometry measuring CD11b markers showed that a high-fat diet-induced neutrophil production in wild-type male mice, but not in wild-type female mice or G0s2 -/- mice, suggesting hormonal regulation involving G0S2 and female mice. Consistent with this observation, forced BCR::ABL1 expression in G0s2 -/- bone marrow reduced overall survival in female (n=4/group) but not male (n=5/group) mice compared with wild-type controls. In mass spectrometry-based lipidomics analyses, G0S2 knockdown resulted in a substantial decrease of di- and triglycerides. G0S2 knockdown also resulted in substantial changes in phosphatidylethanolamine and phosphatidylcholine expression, implicating G0S2 in the production of lipid bilayer components. Conversely, ectopic G0S2 expression promoted the accumulation of long- and very long-chain triglycerides and species of phosphatidylcholine and phosphatidylethanolamine in K562 cells. Proteomics analyses showed autophagy as one of the top pathways affected by altered G0S2 expression. When we measured LC3A/B I-II by immunoblot, we observed opposing effects comparing G0S2 ectopic expression versus knockdown. Conclusion: Altogether, these findings suggest sex-based differences in the role of G0S2 and lipid metabolism in CML stem and progenitor cell differentiation, survival, and autophagy, both in vitro and in vivo. Restoring G0S2 expression combined with BCR::ABL1 inhibition may be a novel clinical strategy to induce treatment-free remission in CML. However, further studies should assess whether this strategy will have similar effects in male versus female patients.

An In Silico Database for Automated Feature Identification of High-Resolution Tandem Mass Spectrometry 13C-Trimethylation Enhancement Using Diazomethane (13C-TrEnDi)-Modified Lipid Data.

13C-Trimethylation enhancement using diazomethane (13C-TrEnDi) is a chemical derivatization technique that uses 13C-labeled diazomethane to increase mass spectrometry (MS) signal intensities for phosphatidylcholine (PC) and phosphatidylethanolamine (PE) lipid classes, both of which are of major interest in biochemistry. In silico mass spectrometry databases have become mainstays in lipidomics experiments; however, 13C-TrEnDi-modified PC and PE species have altered m/z and fragmentation patterns from their native counterparts. To build a database of 13C-TrEnDi-modified PC and PE species, a lipid extract from nutritional yeast was derivatized and fragmentation spectra of modified PC and PE species were mined using diagnostic fragmentation filtering by searching 13C-TrEnDi-modified headgroups with m/z 199 (PC) and 202 (PE). Identities of 25 PC and 10 PE species were assigned after comparing to predicted masses from the Lipid Maps Structure Database with no false positive identifications observed; neutral lipids could still be annotated after derivatization. Collision energies from 16 to 52 eV were examined, resulting in three additional class-specific fragment ions emerging, as well as a combined sn-1/sn-2 fragment ion, allowing sum-composition level annotations to be assigned. Using the Lipid Blast templates, a NIST-compatible 13C-TrEnDi database was produced based on fragmentation spectra observed at 36 eV and tested on HEK 293T cell lipid extracts, identifying 47 PC and 24 PE species, representing a 1.8-fold and 2.2-fold increase in annotations, respectively. The 13C-TrEnDi database is freely available, MS vendor-independent, and widely compatible with MS data processing pipelines, increasing the throughput and accessibility of TrEnDi for lipidomics applications.

Fetal sex differences in placental LCPUFA ether and plasmalogen phosphatidylethanolamine and phosphatidylcholine contents in pregnancies complicated by obesity

BackgroundWe have previously reported that maternal obesity reduces placental transport capacity for lysophosphatidylcholine-docosahexaenoic acid (LPC-DHA), a preferred form for transfer of DHA (omega 3) to the fetal brain, but only in male fetuses. Phosphatidylethanolamine (PE) and phosphatidylcholine (PC), have either sn-1 ester, ether or vinyl ether (plasmalogen) linkages to primarily unsaturated and monounsaturated fatty acids and DHA or arachidonic acid (ARA, omega 6) in the sn-2 position. Whether ether and plasmalogen PC and PE metabolism in placenta impacts transfer to the fetus is unexplored. We hypothesized that ether and plasmalogen PC and PE containing DHA and ARA are reduced in maternal–fetal unit in pregnancies complicated by obesity and these differences are dependent on fetal sex.MethodsIn maternal, umbilical cord plasma and placentas from obese women (11 female/5 male infants) and normal weight women (9 female/7 male infants), all PC and PE species containing DHA and ARA were analyzed by LC–MS/MS. Placental protein expression of enzymes involved in phospholipid synthesis, were determined by immunoblotting. All variables were compared between control vs obese groups and separated by fetal sex, in each sample using the Benjamini–Hochberg false discovery rate adjustment to account for multiple testing.ResultsLevels of ester PC containing DHA and ARA were profoundly reduced by 60–92% in male placentas of obese mothers, while levels of ether and plasmalogen PE containing DHA and ARA were decreased by 51–84% in female placentas. PLA2G4C abundance was lower in male placentas and LPCAT4 abundance was lower solely in females in obesity. In umbilical cord, levels of ester, ether and plasmalogen PC and PE with DHA were reduced by 43–61% in male, but not female, fetuses of obese mothers.ConclusionsWe found a fetal sex effect in placental PE and PC ester, ether and plasmalogen PE and PC containing DHA in response to maternal obesity which appears to reflect an ability of female placentas to adapt to maintain optimal fetal DHA transfer in maternal obesity.

The lipoprotein-associated phospholipase A2 inhibitor Darapladib sensitises cancer cells to ferroptosis by remodelling lipid metabolism

Arachidonic and adrenic acids in the membrane play key roles in ferroptosis. Here, we reveal that lipoprotein-associated phospholipase A2 (Lp-PLA2) controls intracellular phospholipid metabolism and contributes to ferroptosis resistance. A metabolic drug screen reveals that darapladib, an inhibitor of Lp-PLA2, synergistically induces ferroptosis in the presence of GPX4 inhibitors. We show that darapladib is able to enhance ferroptosis under lipoprotein-deficient or serum-free conditions. Furthermore, we find that Lp-PLA2 is located in the membrane and cytoplasm and suppresses ferroptosis, suggesting a critical role for intracellular Lp-PLA2. Lipidomic analyses show that darapladib treatment or deletion of PLA2G7, which encodes Lp-PLA2, generally enriches phosphatidylethanolamine species and reduces lysophosphatidylethanolamine species. Moreover, combination treatment of darapladib with the GPX4 inhibitor PACMA31 efficiently inhibits tumour growth in a xenograft model. Our study suggests that inhibition of Lp-PLA2 is a potential therapeutic strategy to enhance ferroptosis in cancer treatment.

The effect of 2-day heat stress on the lipid composition of bovine milk and serum

Milk and serum were collected from dairy cows before and during a 2-day heat challenge. The concentrations of free short-chain fatty acids (SCFAs), the fatty acid (FA) profile, and the abundance of the major species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM) were measured, and samples collected during heat exposure were compared with those collected prior to heat exposure. It was found that a 2-day heat challenge did not alter the global FA composition of milk fat nor the content of the major phospholipids. Although the concentration of SCFAs C3 and C4 and some lysophosphatidylcholine (LPC) species in milk was found to be associated with the forage type, neither of these lipid molecules can be used as an indicator of acute heat stress. While it is a positive finding that short-term heat stress has no detrimental effect on the FA composition or the nutritive quality of milk fat, this study highlights the complexity of validating a milk lipid biomarker for heat stress in dairy cows.

Green extraction of phospholipids from ghee residue using pulsed electric field processing: Process optimisation and analysis of structural and compositional parameters

Process parameters were optimised using Taguchi orthogonal array design for extraction of phospholipids (PLs) from ghee residue (GR) with pulsed electric field (PEF) assistance. For higher PL yield and antioxidant activity, process voltage and time were observed to be significant factors. Optimised conditions were determined as 60 kV/cm voltage at 7.5 (v/w) solvent (water)‐to‐solid ratio for 5 min. Physical disruption of GR matrix due to PEF treatment was established through scanning electron micrographs. Lipidomic profile of the extract ascertained through liquid chromatography‐mass spectrophotometry (LC–MS) revealed multiple species of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine in the resultant extract.

PE, or not PE, that is the question: The case of overlooked lyso-N-acylphosphatidylethanolamines.

Lyso derivatives of N-acyl-1,2-diacylglycero-3-phosphoethanolamines (L-NAPEs) are a lipid class mostly expressed in vegetables during stress and tissue damage that is involved in the synthesis of the lipid mediator N-acylethanolamines. L-NAPEs can be challenging to distinguish from isomeric phosphatidylethanolamines (PEs), especially in extracted complex samples where they could be confused with abundant PEs. In this study, hydrophilic interaction liquid chromatography with electrospray ionization hyphenated with (tandem) mass spectrometry (MS) was proposed to distinguish L-NAPEs and PEs as deprotonated molecules, [M - H]─ , using both high-resolution/accuracy Fourier transform MS and low-resolution linear ion trap (LIT) mass analyzers. MS3 experiments of [M - H - KE]─ as precursor ions (KE, ketene loss) using the LIT instrument allowed us to distinguish between isomeric L-NAPE and PE species. Regiochemical rules were proposed working on enzymatically synthesized L-NAPEs. A few key differences in MS/MS spectra, including abnormal intensity of acyl chain losses as fatty acids, the presence of N-acylphosphoethanolamine ions, and diagnostic ions of the polar head, were disclosed. Additionally, MS3 spectra of [M - H - KE]─ as precursor ions allowed us to confirm the identification of L-NAPE species. The proposed rules were applied to samples extracted from tomato by-products including stems and leaves. Overall, our methodology is demonstrated as a robust approach to recognizing L-NAPEs in complex samples. L-NAPEs 18:2-N-18:2, 18:2-N-18:3, 18:3-N-18:2, and 18:2-N-18:1 were the prevailing compounds in the analyzed tomato samples, accounting for more than 90%. In summary, a reliable method for identifying L-NAPEs in complex samples is described. The proposed method could prevent overlooking L-NAPEs and overestimating isomeric PE species in future lipid analyses.

Red blood cells from patients with sitosterolemia exhibit impaired membrane lipid composition and distribution and decreased deformability

Sitosterolemia is a metabolic disorder leading to excessive accumulation of phytosterols. Hemolytic stomatocytosis and macrothrombocytopenia are part of the clinical picture. However, the impact of phytosterols on red blood cell (RBC) deformability, membrane lipid composition and distribution and the efficiency of the reference treatment, Ezetimibe, are largely unknown. This study addresses these issues using RBCs from three patients with sitosterolemia and healthy RBCs exposed to β-sitosterol. Patients presented an increased proportion of stomatocytes, decreased RBC deformability and increased RBC hydration and osmotic fragility compared to healthy donors. At the membrane level, patient RBCs showed (i) very high content in β-sitosterols, (ii) increased proportions of saturated fatty acids and polyunsaturated fatty acid species with long and unsaturated carbon chains, and (iii) decreased content in phosphatidylethanolamine species. These lipid changes were accompanied by an almost complete abrogation of cholesterol-enriched domains, which could result from: (i) the reduced phosphatidylethanolamine content which positively correlated with domain abundance; and (ii) the fatty acid modifications and increased phytosterol content, both compatible with higher membrane stiffness. The role of β-sitosterol was supported by comparable changes in RBC morphology and cholesterol-enriched domains upon β-sitosterol integration at the healthy RBC membrane. Finally, Ezetimibe treatment combined with a sterol restricted diet lowered phytosterols and improved anemia and RBC deformability and hydration. However, this treatment had no or limited effect on RBC morphology and cholesterol-enriched domain abundance. This study reveals for the first time that phytosterols affect RBC membrane lipid composition and distribution but also RBC morphology, hydration, deformability and fragility.

Redox phospholipidomics discovers pro-ferroptotic death signals in A375 melanoma cells in vitro and in vivo

Growing cancer cells effectively evade most programs of regulated cell death, particularly apoptosis. This necessitates a search for alternative therapeutic modalities to cause cancer cell's demise, among them – ferroptosis. One of the obstacles to using pro-ferroptotic agents to treat cancer is the lack of adequate biomarkers of ferroptosis. Ferroptosis is accompanied by peroxidation of polyunsaturated species of phosphatidylethanolamine (PE) to hydroperoxy- (-OOH) derivatives, which act as death signals. We demonstrate that RSL3-induced death of A375 melanoma cells in vitro was fully preventable by ferrostatin-1, suggesting their high susceptibility to ferroptosis. Treatment of A375 cells with RSL3 caused a significant accumulation of PE-(18:0/20:4-OOH) and PE-(18:0/22:4-OOH), the biomarkers of ferroptosis, as well as oxidatively truncated products - PE-(18:0/hydroxy-8-oxo-oct-6-enoic acid (HOOA) and PC-(18:0/HOOA). A significant suppressive effect of RSL3 on melanoma growth was observed in vivo (utilizing a xenograft model of inoculation of GFP-labeled A375 cells into immune-deficient athymic nude mice). Redox phospholipidomics revealed elevated levels of 18:0/20:4-OOH in RSL3-treated group vs controls. In addition, PE-(18:0/20:4-OOH) species were identified as major contributors to the separation of control and RSL3-treated groups, with the highest variable importance in projection predictive score. Pearson correlation analysis revealed an association between tumor weight and contents of PE-(18:0/20:4-OOH) (r = −0.505), PE-18:0/HOOA (r = −0.547) and PE 16:0-HOOA (r = −0.503). Thus, LC-MS/MS based redox lipidomics is a sensitive and precise approach for the detection and characterization of phospholipid biomarkers of ferroptosis induced in cancer cells by radio- and chemotherapy.

P068 Polyunsaturated fatty acid excess fuels pro-inflammatory transcriptional networks in GPX4 deficient intestinal epithelial cells

Abstract Background There is increasing evidence that the rise in Crohn’s disease (CD) incidence is partially driven by constituents of a Western-style diet such as polyunsaturated fatty acids (PUFA). Recently we could demonstrate that PUFA excess induces a CD-like enteritis in mice lacking antioxidant GPX4 in their intestinal epithelial cells (IECs) and that CD patients clinical disease activity positively correlates with PUFA intake. In this project we investigated the role of epithelial lipid composition and lipid mediators during PUFA induced metabolic gut inflammation. Furthermore, we transcriptionally profiled GPX4 deficient small intestinal cells after stimulation with ω3- and ω6-PUFA. Methods Mice lacking one allele of Gpx4 in their IECs (GPX4+/-IEC) and wild-type controls (WT) were fed a PUFA (10% fish oil) enriched Western diet (PUFA-WD) for 12 weeks. Intestinal inflammation was assessed by histopathology. IECs were harvested by scraping of small intestinal sections and lipid profiles and mediators were analysed with LC/MS. GPX4 in murine small intestinal cells (MODE-K) was knocked down by siRNA (siGpx4) scrambled siRNA served as control (siCtrl). MODE-K were stimulated with ω3-, ω6-PUFA, 5,6- and 8,9-DHET or vehicle. Cytokine expression in cell culture supernatant was analysed by ELISA and transcriptome was analysed by bulk RNA sequencing. Results Histopathology revealed gut inflammation in GPX4+/-IEC but not WT mice after 12 weeks PUFA-WD feeding. LC/MS analysis of IECs derived from GPX4+/-IEC and WT mice fed the PUFA-WD depicted a significant increase in eicosapentaenoic acid (20:5), docosapentaenoic acid (22:5) and docosahexaenoic acid (22:6) containing phosphatidylethanolamine (PE) and phosphatidylcholine (PC) species (Fig 1a). All other PE and PC species showed a reduced amount, confirming the enrichment of the specific PUFAs within the IECs by the PUFA-WD. Lipid mediator analysis further revealed an increase in 5,6-DHET and 8,9-DHET only in IECs derived from GPX4+/-IEC but not WT mice (Fig 1b). When stimulated with PUFA, siGpx4 MODE-K produced high amounts of neutrophil attracting chemokine CXCL-1 which is attenuated by the supplementation of anti-inflammatory lipid mediators 5,6-DHET and 8,9-DHET (Fig 2). Transcriptome analysis revealed the upregulation of pro-inflammatory pathways including TNFa, MAPK and NF-κB in siGpx4 vs. siCtrl MODE-K after PUFA stimulation (Fig 3). Conclusion By integrating lipidomic and transcriptomic data of a reductionist cell culture system and a model organism, we could show that PUFA excess in a GPX4 deficient individual fuels a pro-inflammatory transcriptional network which outweighs anti-inflammatory ω3-PUFA metabolites influence culminating in gut inflammation.

Modulations of bioactive lipids and their receptors in postmortem Alzheimer's disease brains.

Analyses of brain samples from Alzheimer's disease (AD) patients may be expected to help us improve our understanding of the pathogenesis of AD. Bioactive lipids, including sphingolipids, glycerophospholipids, and eicosanoids/related mediators have been demonstrated to exert potent physiological actions and to be involved in the pathogenesis of various human diseases. In this cross-sectional study, we attempted to elucidate the associations of these bioactive lipids with the pathogenesis/pathology of AD through postmortem studies of human brains. We measured the levels of glycerophospholipids, sphingolipids, and eicosanoids/related mediators in the brains of patients with AD (AD brains), patients with Cerad score B (Cerad-b brains), and control subjects (control brains), using a liquid chromatography-mass spectrometry method; we also measured the mRNA levels of specific receptors for these bioactive lipids in the same brain specimens. The levels of several species of sphingomyelins and ceramides were higher in the Cerad-b and AD brains. Levels of several species of lysophosphatidic acids (LPAs), lysophosphatidylcholine, lysophosphatidylserine, lysophosphatidylethanolamine (LPE), lysophosphatidylinositol, phosphatidylcholine, phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol, and phosphatidylglycerol were especially high in the Cerad-b brains, while those of lysophosphatidylglycerol (LPG) were especially high in the AD brains. Several eicosanoids, including metabolites of prostaglandin E2, oxylipins, metabolites of epoxide, and metabolites of DHA and EPA, such as resolvins, were also modulated in the AD brains. Among the lipid mediators, the levels of S1P2, S1P5, LPA1, LPA2, LPA6, P2Y10, GPR174, EP1, DP1, DP2, IP, FP, and TXA2r were lower in the AD and/or Cerad-b brains. The brain levels of ceramides, LPC, LPI, PE, and PS showed strong positive correlations with the Aβ contents, while those of LPG showed rather strong positive correlations with the presence of senile plaques and neurofibrillary tangles. A discriminant analysis revealed that LPG is especially important for AD and the LPE/PE axis is important for Cerad-b. Comprehensive lipidomics, together with the measurement of lipid receptor expression levels provided novel evidence for the associations of bioactive lipids with AD, which is expected to facilitate future translational research and reverse translational research.

Abstract 13353: Association of Plasma Metabolites With Inflammatory Markers CRP and GlycA and Their Responses to Exercise Training

Introduction. C-reactive protein (CRP) and GlycA are established biomarkers of inflammation. Regular exercise tends to decrease CRP and GlycA levels. However, the spectrum of molecules associated with the anti-inflammatory effects of regular exercise are less well understood. Hypothesis. We hypothesized that distinct metabolite signatures exist for both baseline levels and exercise responsiveness of CRP and GlycA. Methods. Measures were performed before and after 20 weeks of endurance exercise training in 652 Black and White adults from the HERITAGE Family Study. A total of 300 targeted plasma metabolites were measured using LC-MS. High-sensitivity CRP and GlycA were measured using automated assays and NMR spectroscopy (LabCorp), respectively. Linear mixed models were used to test: 1) Association of baseline metabolites with baseline hsCRP and GlycA and 2) Association of changes in metabolite with changes in hsCRP and GlycA. Models were adjusted for age, sex, race, BMI, with family membership as a random variable, with change models also adjusting for baseline trait value. Significance was determined as FDR<0.05. Results. Baseline levels and changes of hsCRP and GlycA were moderately correlated (r=0.51 and 0.31, p<0.0001 for both, respectively). At baseline, 40 and 94 metabolites were associated with hsCRP and GlycA, respectively, with 30 metabolites associated with both phenotypes. The top baseline associations for both traits included multiple species of lysophosphatidylcholine (LPC) and phosphatidylethanolamine (PE), while cortisol, biliverdin, and bilirubin were among the metabolites associated with GlycA only. The changes in only one metabolite were associated with concomitant changes in CRP, while no associations were found for change in GlycA. Conclusions. Plasma metabolite associations with baseline hsCRP overlapped with those associated with baseline GlycA levels. Several unique metabolite associations with baseline GlycA were identified, including molecules in established inflammatory pathways. Metabolite changes with exercise were not associated with changes in either measure. These findings have implications for the use of metabolites as signatures of systemic inflammation vs as targets of lifestyle interventions.

Light modulates the lipidome of the photosynthetic sea slug Elysia timida

Long-term kleptoplasty, the capability to retain functional stolen chloroplasts (kleptoplasts) for several weeks to months, has been shown in a handful of Sacoglossa sea slugs. One of these sea slugs is Elysia timida, endemic to the Mediterranean, which retains functional chloroplasts of the macroalga Acetabularia acetabulum. To understand how light modulates the lipidome of E. timida, sea slug specimens were subjected to two different 4-week light treatments: regular light and quasi-dark conditions. Lipidomic analyses were performed by HILIC-HR-ESI-MS and MS/MS. Quasi-dark conditions caused a reduction in the amount of essential lipids for photosynthetic membranes, such as glycolipids, indicating high level of kleptoplast degradation under sub-optimal light conditions. However, maximum photosynthetic capacities (Fv/Fm) were identical in both light treatments (≈0.75), showing similar kleptoplast functionality and suggesting that older kleptoplasts were targeted for degradation. Although more stable, the phospholipidome showed differences between light treatments: the amount of certain lipid species of phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylglycerol (PG) decreased under quasi-dark conditions, while other lipid species of phosphatidylcholine (PC), PE and lyso-PE (LPE) increased. Quasi-dark conditions promoted a decrease in the relative abundance of polyunsaturated fatty acids. These results suggest a light-driven remodelling of the lipidome according to the functions of the different lipids and highlight the plasticity of polar lipids in the photosynthetic sea slug E. timida.