Phosphatidylcholine Species Research Articles

Quantitative determination of sn-positional phospholipid isomers in MSn using silver cationization

Glycerophospholipids are one of the fundamental building blocks for life. The acyl chain connectivity to the glycerol backbone constitutes different sn-positional isomers, which have great diversity and importance for biological function. However, to fully realize their impact on function, analytical techniques that can identify and quantify sn-positional isomers in chemically complex biological samples are needed. Here, we utilize silver ion cationization in combination with tandem mass spectrometry (MSn) to identify sn-positional isomers of phosphatidylcholine (PC) species. In particular, a labile carbocation is generated through a neutral loss (NL) of AgH, the dissociation of which provides diagnostic product ions that correspond to acyl chains at the sn-1 or sn-2 position. The method is comparable to currently available methods, has a sensitivity in the nM–µM range, and is compatible with quantitative imaging using mass spectrometry in MS4. The results reveal a large difference in isomer concentrations and the ion images show that the sn-positional isomers PC 18:1_18:0 are homogeneously distributed, whereas PC 18:1_16:0 and PC 20:1_16:0 show distinct localizations to sub-hippocampal structures.Graphical abstract

Infusion Mass Spectrometric Analysis of Sphingomyelin Species in Dairy With Lithium-Mediated Fragmentation

ObjectivesBovine milk is rich in sphingomyelin (SM), a class of polar lipids which may affect the gut microbiome and have anti-inflammatory properties. Infusion mass spectrometry rapidly yields valuable information about the structure of SM, but quantitation is confounded by isobaric phosphatidylcholine (PC) lipids. Using previously established Li-induced fragmentation chemistry, we developed a quantitative method for analysis of SM species using selective multiple reaction monitoring (MRM) transitions.MethodsPolar lipids were extracted from milk samples using organic solvents followed by silicic acid solid phase extraction (SPE) to remove triacylglycerol (TAG). Extracts were dissolved in methanol with 0.2 mM lithium acetate and analyzed on an AB Sciex 5500 QTRAP hybrid mass spectrometer in an infusion-based manner. SM species were identified by a characteristic 207 Da loss, representing the loss of [Li + H2O + (CH3)3NC2H4PO4] allowing for separation from PC species. Commercially available SM standards were used where possible to verify identity. A total SM profile was determined through neutral loss (NL) 207 Da scans, SM fine structure with MS3 scans, and individual SM species were quantified utilizing MRM.ResultsUse of the Li- mediated fragmentation and the NL 207 Da scans allowed for selective quantitation of SM, but not PC species, with limits of detection >1 nM and limits of quantitation >10 nM for individual SM species. Milk matrix TAG suppressed MRM intensities by 10-fold requiring SPE removal of TAG. In bovine milk, 30 individual SM species were detected ranging from SM 28:1 to SM 44:2. SM were predominately 16:1 and 18:1 sphingoid bases with fatty acid tails ranging from 10 to 26 carbons. Utilizing this method, we determined the SM content of fluid dairy products including whole bovine milk (37 μM), half and half (70 μM), whipping cream (130 μM), and goat milk (56 μM).ConclusionsA rapid, infusion-based MS approach was developed to selectivity identify and quantify SM species that avoids isobaric overlap with PC. This method is robust and could potentially be applied to other SM-containing samples.Funding SourcesThis work was funded by USDA-ARS Projects 3062-53,000-001-00D and supported by the USDA-ARS Grand Challenge Synergy project “Dairy Agriculture for People and Planet”.

The Effects of Wolfberry Consumption on the Human Plasma Lipidome and Its Potential Role in Cardiovascular Disease Risk Modulation: A Randomized Controlled Trial

ObjectivesThe adverse effects of dyslipidemia on cardiovascular health are well-documented. Conventionally assayed by blood lipids/lipoproteins, these classical indicators alone yield an oversimplified view of the human plasma lipidome. We previously observed an attenuation of cardiovascular disease (CVD) risk after regular wolfberry consumption for 16 weeks, with a prominent reduction in oxidative stress. To complement these findings, the objective of this study was to characterize the alterations to the plasma lipidome and explore its potential correlations to CVD risk. MethodsIn this parallel randomized controlled trial, middle-aged and older adults (55 ± 4 y) from Singapore were counselled to follow a healthy dietary pattern, either with or without consuming 15 g cooked whole wolfberry daily (WB; n = 21, CON; n = 17). Following lipid extraction of weeks 0 and 16 plasma samples, the lipidome was assayed by high-performance liquid chromatography-tandem mass spectrometry and quantified with internal standards (427 lipid species). Pearson’s correlations between the fold changes (FCs) of lipid species from baseline, with corresponding changes to CVD risk-related outcomes including blood lipids/lipoproteins, blood pressure as well as vascular health and oxidative stress-related indicators were further evaluated. ResultsA comparison between the groups identified 7 distinct lipid species between WB and CON (FC > 1.2; P < 0.05). Notably, sphingomyelin 38:1, which had a markedly higher FC in WB compared to CON (FCWB: 1.2, FCCON: 1.0), was also significantly raised compared to baseline. Phosphatidylcholine (PC) 35:2, which increased by 1.8-fold in WB only (P < 0.05), additionally depicted an inverse linear correlation with the FC of plasma 8-isoprostanes (Pearson’s correlation coefficient: –0.45, P < 0.05). Across both groups, FCs from baseline were also significant for species of triglycerides (TG; 42 species), PC (4 species), ether-TG, ether-PC, plasmalogen-PC and cholesteryl ester (1 species each) albeit independent of wolfberry intake and with no clear associations to the other CVD risk-related outcomes. ConclusionsCharacteristic alterations to the plasma lipidome observed with wolfberry consumption may potentially modulate its underlying antioxidant effect. Funding SourcesMinistry of Education, Singapore

Phospholipids modifications, genotoxic and anticholinesterase effects of pepper fruit (Dennettia tripetala G. Baker) extract in Swiss mice

The toxicity of D. tripetala fruit extract to mice was investigated using data obtained from lipidomic analyses, comet and Acetylcholinesterase (AChE) assays. Mice (n = 8) were exposed for 30 days via oral gavage to vehicle (5% Tween 80) (negative control), D. tripetala extract (100, 200 and 400 mg/kg) and 40 mg/kg methyl methanesulfonate (MMS) (positive control). The profile of compounds in the fruit extract was analyzed using gas chromatography-mass spectrometry. Out of the total of 32 compounds identified, considerable amount of established insecticidal compounds such as 2-phenylnitroethane, cis-vaccenic acid, linalool and linoleic acid were detected. Fruit extract did not induce DNA damage relative to negative control. Percentage gain in body weights differed significantly across the four weeks. Significantly highest and lowest brain AChE activity was observed in animals exposed to 200 and 400 mg/kg D. tripetala, respectively. Fruit extract modulated the brain phospholipid profile due to significant fold changes of 48 lipid species out of the total of 280 lipid species. High number of differentially expressed phosphatidylcholine (PC) species and significant levels of phosphatidylethanolamine (PE) at 400 mg/kg suggests that activation of inflammation and methylation pathways are the most plausible mechanisms of D. tripetala toxicity to mouse brain tissue.

An Association Between Saturated Fatty Acid-Containing Phosphatidylcholine in Cerebrospinal Fluid with Tau Phosphorylation.

Mechanistic studies in animal models implicate a role for saturated fatty acids in neurodegeneration, but validation of this finding in human studies is still lacking. We investigated how cerebrospinal levels of sphingomyelins (SM) and phosphatidylcholine (PC)-containing saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids associate with total tau and phosphorylated tau (p-tau). Cerebrospinal fluid (CSF) lipids were measured in two cohorts, a discovery and a confirmation cohort of older non-demented individuals from the University of Southern California and Huntington Medical Research Institutes cohorts. Lipid analysis was performed using hydrophilic interaction liquid chromatography, and individual PC and SM lipid species were measured using tandem mass spectrometry. In addition, CSF levels of Aβ42, total tau, and p-tau-181 were measured using an MSD multiplex assay. The discovery cohort (n = 47) consisted of older individuals and more females compared to the confirmation cohort (n = 46). Notwithstanding the age and gender differences, and a higher p-tau, Aβ42, and LDL-cholesterol in the discovery cohort, CSF concentrations of dipalmitoyl-PC (PC32a:0) were significantly associated with p-tau in both cohorts. Similarly, total saturated PC but not mono or polyunsaturated PCs correlated with p-tau concentrations in both cohorts. Saturated PC species in CSF associate with early markers of neurodegeneration and are potential early disease progression biomarkers. We propose mechanisms by which saturated PC may promote tau hyperphosphorylation.

Lipid pathway dysfunction is prevalent in patients with Parkinson's disease.

Many genetic risk factors for Parkinson’s disease have lipid-related functions and lipid-modulating drugs such as statins may be protective against Parkinson’s disease. Moreover, the hallmark Parkinson’s disease pathological protein, α-synuclein, has lipid membrane function and pathways dysregulated in Parkinson’s disease such as the endosome–lysosome system and synaptic signalling rely heavily on lipid dynamics. Despite the potential role for lipids in Parkinson’s disease, most research to date has been protein-centric, with large-scale, untargeted serum and CSF lipidomic comparisons between genetic and idiopathic Parkinson’s disease and neurotypical controls limited. In particular, the extent to which lipid dysregulation occurs in mutation carriers of one of the most common Parkinson’s disease risk genes, LRRK2, is unclear. Further, the functional lipid pathways potentially dysregulated in idiopathic and LRRK2 mutation Parkinson’s disease are underexplored.To better determine the extent of lipid dysregulation in Parkinson’s disease, untargeted high-performance liquid chromatography–tandem mass spectrometry was performed on serum (n = 221) and CSF (n = 88) obtained from a multi-ethnic population from the Michael J. Fox Foundation LRRK2 Clinical Cohort Consortium. The cohort consisted of controls, asymptomatic LRRK2 G2019S carriers, LRRK2 G2019S carriers with Parkinson’s disease and Parkinson’s disease patients without a LRRK2 mutation. Age and sex were adjusted for in analyses where appropriate. Approximately 1000 serum lipid species per participant were analysed.The main serum lipids that distinguished both Parkinson’s disease patients and LRRK2 mutation carriers from controls included species of ceramide, triacylglycerol, sphingomyelin, acylcarnitine, phosphatidylcholine and lysophosphatidylethanolamine. Significant alterations in sphingolipids and glycerolipids were also reflected in Parkinson’s disease and LRRK2 mutation carrier CSF, although no correlations were observed between lipids identified in both serum and CSF. Pathway analysis of altered lipid species indicated that sphingolipid metabolism, insulin signalling and mitochondrial function were the major metabolic pathways dysregulated in Parkinson’s disease. Importantly, these pathways were also found to be dysregulated in serum samples from a second Parkinson’s disease cohort (n = 315).Results from this study demonstrate that dysregulated lipids in Parkinson’s disease generally, and in LRRK2 mutation carriers, are from functionally and metabolically related pathways. These findings provide new insight into the extent of lipid dysfunction in Parkinson’s disease and therapeutics manipulating these pathways may be beneficial for Parkinson’s disease patients. Moreover, serum lipid profiles may be novel biomarkers for both genetic and idiopathic Parkinson’s disease.

Unusual Lipid Components of Legionella gormanii Membranes.

Legionella spp. cause Legionnaires’ disease with pneumonia as the predominant clinical symptom. L. gormanii is the second most prevalent causative agent of community-acquired pneumonia after L. pneumophila. The study aimed to characterize the lipidome of L. gormanii membranes and the importance of these analyses in bacterial chemotaxonomy. Lipidomic analyses based on ultra-high performance liquid chromatography-mass spectrometry allowed the detection of individual molecular species of a wide range of L. gormanii membrane lipids contained in the outer (OM) and inner membranes (IM). The lipid profile comprised glycerolipids (triglycerides, diglycerides), phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, cardiolipin), and sphingolipids (ceramides, hexosylceramides). The most abundant lipid fraction in the IM and OM were phospholipids. The lipidomic analysis showed that two independent phosphatidylcholine (PC) synthesis pathways operating in L. gormanii: the PE-methylation (PmtA) pathway and the PC synthase (Pcs) pathway. Comparison of the molecular profile of PC species contained in the lipids of L. gormanii membranes cultured on the medium, with and without exogenous choline, showed quantitative differences in the PC pool. An unusual feature of the L. gormanii lipids was the presence of ceramides and hexosylceramides, which are typical components of eukaryotic cells and a very small group of bacteria. To the best of our knowledge, this is the first report of the occurrence of ceramides in Legionella bacteria.

The Acyltransferase Gpc1 is Both a Target and an Effector of the Unfolded Protein Response (UPR)

The unfolded protein response (UPR) is sensitive to both proteotoxic stress and membrane bilayer stress. These stresses are sensed by the ER transmembrane protein Ire1. When activated, Ire1 uses its endonuclease activity to splice HAC1mRNA, producing a mature transcription factor that binds to UPR elements (UPREs) in the promoters of target genes. Hac1 targets include not only genes involved in protein folding, secretion, and degradation, but also a subset of lipid metabolic genes. One aspect of lipid metabolism is the deacylation of phosphatidylcholine (PC) by phospholipases to produce glycerophosphocholine (GPC). In Saccharomyces cerevisiae, GPC can be reacylated in a novel two‐step process catalyzed first by GPC acyltransferase Gpc1, followed by acylation of the lyso‐PC molecule by Ale1. This metabolic cycle has been termed the PC deacylation/reacylation pathway (PC‐DRP). In prior studies, loss of Gpc1 was shown to result in an increase in di‐unsaturated PC species at the expense of mono‐unsaturated PC species, indicating a role for PC‐DRP in PC acyl chain remodeling. Here, we probe the role of Gpc1 as both a target and an effector of the UPR. Exposure to the UPR‐inducing compounds tunicamycin, DTT, and canavanine results in an increase in GPC1 message that is dependent upon the UPR transcriptional activator Hac1. The importance of this increased expression to cellular function is illustrated by the finding that cells lacking Gpc1 exhibit increased sensitivity to those compounds. In a converse set of experiments, we show that that loss of GPC1 results in upregulation of the UPR as measured by expression of the ER chaperone KAR2. Consistent with these findings, we show that Gpc1 primarily co‐localizes with the endoplasmic reticulum.

Impact of Dietary Empagliflozin on Renal Cortex Metabolome and Lipidome in Mice

Sodium glucose cotransporter, type 2 inhibitors (SGLT2i) have been demonstrated to be protective of kidney by unclear mechanisms. The aim of the current study was to determine how treatment with a common SGLT2i, Empagliflozin affected kidney cortical metabolome and lipidome in high‐fat‐fed, male TallyHo/Jng (TH) mice. TH mice are prone to obesity and insulin resistance progressing to type 2 diabetes (T2D). Adult mice (n = 7 or 8/group) were fed a control high‐milk‐fat diet (60% by weight) or this same diet supplemented with empagliflozin (0.01%) for 8‐weeks, then euthanized and kidney cortex dissected and rapidly frozen. Metabolomics (targeted and untargeted) and lipidomics (targeted) were conducted on separate sections of cortex in our core facility by liquid chromatography‐followed by tandem mass‐spectroscopy. Data pre‐processing included signal drift and inter‐batch correction, as well as, removal of species with high coefficient of variability on pooled or reference analysis. Metabolites and lipids passing quality control (QC) included 207 and 467 unique species, respectively, which were further analyzed by MetaboAnalyst 5.0 and lipids, additionally by LipidSig, both publically available software platforms. Volcano plotting of metabolites and lipids revealed oppositely skewed patterns in that metabolites tended to be relatively decreased and lipid species increased with Empagliflozin. Seventy‐four metabolites met our threshold of changed &gt; 20% change and/or p &lt; 0.05 and were analyzed for pathway enrichment. The top three pathways down regulated by Empagliflozin were urea cycle, spermine/spermidine biosynthesis, and aspartate metabolism. Analysis of metabolites of glycolysis/oxidative phosphorylation revealed a general 20‐45% reduction in several species including phosphoenolpyruvate (PEP), succinate, and malic acid. In contrast, in general, several lipid species were increased with hierarchical clustering showing greatest effect on phosphatidylglycerol (16:1/16:1), 40% increase, p &lt; 0.02, lysophosphatidylcholine (LPC, 20:4) 38% increase p &lt; 0.03, and 10 additional phosphatidylcholine (PC) species. Overall, these analyses suggest a greater abundance of lipid, relative to carbohydrate and protein‐based species, in the kidney cortex in response to chronic Empagliflozin treatment. It is unclear whether these differences are due to intracellular metabolic alterations or transport reduction in substrate (glucose and peptides) for metabolic pathways. In addition, we found no evidence of elevated gluconeogenesis with Empagliflozin observing a significant reduction in one key metabolite in this particular pathway, PEP. We may speculate oxidative phosphorylation and ATP generation is maintained relatively normal by greater oxidation of lipids (as opposed to proteins and carbohydrates) in the kidney proximal tubule of Empagliflozin‐treated mice.

Lipids associated with plant-bacteria interaction identified using a metabolomics approach in an Arabidopsis thaliana model.

BackgroundSystemic acquired resistance (SAR) protects plants against a wide variety of pathogens. In recent decades, numerous studies have focused on the induction of SAR, but its molecular mechanisms remain largely unknown.MethodsWe used a metabolomics approach based on ultra-high-performance liquid chromatographic (UPLC) and mass spectrometric (MS) techniques to identify SAR-related lipid metabolites in an Arabidopsis thaliana model. Multiple statistical analyses were used to identify the differentially regulated metabolites.ResultsNumerous lipids were implicated as potential factors in both plant basal resistance and SAR; these include species of phosphatidic acid (PA), monogalactosyldiacylglycerol (MGDG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and triacylglycerol (TG).ConclusionsOur findings indicate that lipids accumulated in both local and systemic leaves, while other lipids only accumulated in local leaves or in systemic leaves. PA (16:0_18:2), PE (34:5) and PE (16:0_18:2) had higher levels in both local leaves inoculated with Psm ES4326 or Psm avrRpm1 and systemic leaves of the plants locally infected with Psm avrRpm1 or Psm ES4326. PC (32:5) had high levels in leaves inoculated with Psm ES4326. Other differentially regulated metabolites, including PA (18:2_18:2), PA (16:0_18:3), PA (18:3_18:2), PE (16:0_18:3), PE (16:1_16:1), PE (34:4) and TGs showed higher levels in systemic leaves of the plants locally infected with Psm avrRpm1 or Psm ES4326. These findings will help direct future studies on the molecular mechanisms of SAR.

Controlled Tetradeuteration of Straight-Chain Fatty Acids: Synthesis, Application, and Insight into the Metabolism of Oxidized Linoleic Acid.

We describe a concise and reliable protocol for the precisely controlled tetradeuteration of straight‐chain fatty acids (FAs) at the α‐ and β‐positions that is generally applicable to a variety of FAs, including trans‐FAs, polyunsaturated FAs (PUFAs), and their oxidized derivatives. The precisely controlled introduction of four deuterium atoms into the FAs enables their persistent and quantitative tracking by LC‐MS/MS analysis based on their molecular structures. In addition, the phosphatidylcholine (PC) species prepared from the tetradeuterated FAs thus obtained give a diagnostic peak, namely, a phosphocholine fragment that contains deuterium, in the LC‐MS/MS analysis. With these features, the metabolism of a representative oxidized linoleic acid, that is, hydroxyoctadecadienoic acid (HODE), was investigated, leading to the identification of acyltransferases that transfer the acyl moiety derived from HODE to lysophosphatidylcholine.

Controlled Tetradeuteration of Straight‐Chain Fatty Acids: Synthesis, Application, and Insight into the Metabolism of Oxidized Linoleic Acid

AbstractWe describe a concise and reliable protocol for the precisely controlled tetradeuteration of straight‐chain fatty acids (FAs) at the α‐ and β‐positions that is generally applicable to a variety of FAs, including trans‐FAs, polyunsaturated FAs (PUFAs), and their oxidized derivatives. The precisely controlled introduction of four deuterium atoms into the FAs enables their persistent and quantitative tracking by LC‐MS/MS analysis based on their molecular structures. In addition, the phosphatidylcholine (PC) species prepared from the tetradeuterated FAs thus obtained give a diagnostic peak, namely, a phosphocholine fragment that contains deuterium, in the LC‐MS/MS analysis. With these features, the metabolism of a representative oxidized linoleic acid, that is, hydroxyoctadecadienoic acid (HODE), was investigated, leading to the identification of acyltransferases that transfer the acyl moiety derived from HODE to lysophosphatidylcholine.

Patterns of alpha-linolenic acid incorporation into phospholipids in H4IIE cells

Alpha linolenic acid (ALA) is an 18-carbon essential fatty acid found in plant-based foods and oils. While much attention has been placed on conversion of ALA to long chain polyunsaturated fatty acids, alternative routes of ALA metabolism exist and may lead to formation of other bioactive metabolites of ALA. The current study employed a non-targeted metabolomics approach to profile ALA metabolites that are significantly upregulated by ALA treatment. H4IIE hepatoma cells (n=3 samples per time point) were treated with 60 μM ALA or vehicle for 0, 0.25, 0.5, 1, 2, 3, 4, 6, 8, and 12 h. Samples were then extracted with methanol and analyzed using high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry. We observed selective changes in ALA incorporation into phospholipid classes and subclasses over the 12 h following ALA treatment. While levels of specific molecular species of ALA-containing phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and lysophospholipids were elevated with ALA treatment, others were not affected. Of the phospholipids that were increased, some (e.g., PC[18:3/18:1], PC[18:3/18:4], PE[18:3/18:2], PE[18:3/18:3]) were elevated almost immediately after exposure to ALA, while others [e.g., PE[18:1/18:3] PA[18:3/22:6], and PA[18:3/18:2]) were not elevated until several hours after ALA treatment. Overall, these results suggest that ALA incorporation into phospholipids is selective and support a metabolic hierarchy for ALA incorporation into specific phospholipids. Given the functionality of phospholipids based on their fatty acid composition, future studies will need to investigate the implications of ALA incorporation into specific phospholipids on cell function.

Investigation of oyster Crassostrea gigas lipid profile from three sea areas of China based on non-targeted lipidomics for their geographic region traceability

The present study sought to analyze the lipid profiles of oyster Crassostrea gigas from Yellow Sea (YS), East China Sea, and South China Sea (SCS) through the untargeted lipidomics strategy based on UPLC-Q-Exactive Orbitrap mass spectrometry and multivariate statistics. The results elucidated that geographical differences had profound effects on the lipid content, composition, and lipid molecular profiles. Notably, oysters from the YS group contained the highest lipid content, including triacylglycerol, diacylglycerols, and the majority of phospholipid molecule species, while oysters from the ECS group contained most of the phosphatidylcholine species and the SCS group contained most of the sphingolipid species. Totally, 1155 lipid molecular species belonging to 21 subclasses were identified; of them, 45 lipid molecular species could serve as differential marker for lipid of oysters from different sea areas. Overall, lipidomics could be a potential approach for discrimination of lipid characters between marine shellfishes for geographical origin traceability.

The human LL-37 peptide exerts antimicrobial activity against Legionella micdadei interacting with membrane phospholipids

Legionella micdadei is responsible for community- or nosocomial-acquired pneumonia as well as the influenza-like illness Pontiac fever. The aim of this study was to investigate the ability of L. micdadei to utilize extracellular choline for phosphatidylcholine (PC) synthesis and its consequences for the phospholipid composition of its membrane system and the interaction with the human LL-37 peptide. Comparative analysis of the PC content using isotopic labeling revealed that in presence of exogenous choline 98% of the total PC was synthesized via the Pcs pathway while the remaining 2% were generated via the PE-methylation (PmtA) pathway. PC species were to a greater extent defined by the Pcs pathway in the outer membrane than in the inner membrane. While no major changes in the bacterial lipid content were observed using 31P NMR, indication for utilization of longer acyl chains and slight increase of PG in response to choline addition was observed by a top-down lipidomics screen. The LL-37 peptide inhibited L. micdadei growth in a dose-dependent manner. Bacteria cultured with exogenous choline were more sensitive to the LL-37 peptide when compared to the standard culture condition. Our biophysical investigations show that the peptide perturbs bacterial-derived phospholipid monolayers and this interaction is dependent on the molar portion of PC. This interaction is responsible for the observed changes in the anti-L. micdadei activity of the LL-37 peptide.

The lipid transporter Mfsd2a maintains pulmonary surfactant homeostasis

Pulmonary surfactant is a lipoprotein complex essential for lung function, and insufficiency or altered surfactant composition is associated with major lung diseases, such as acute respiratory distress syndromes, idiopathic pulmonary fibrosis, and chronic obstructive pulmonary disease. Pulmonary surfactant is primarily composed of phosphatidylcholine (PC) in complex with specialized surfactant proteins and secreted by alveolar type 2 (AT2) cells. Surfactant homeostasis on the alveolar surface is balanced by the rates of synthesis and secretion with reuptake and recycling by AT2 cells, with some degradation by pulmonary macrophages and loss up the bronchial tree. However, whether phospholipid (PL) transporters exist in AT2 cells to mediate reuptake of surfactant PL remains to be identified. Here, we demonstrate that major facilitator superfamily domain containing 2a (Mfsd2a), a sodium-dependent lysophosphatidylcholine (LPC) transporter, is expressed at the apical surface of AT2 cells. A mouse model with inducible AT2 cell–specific deficiency of Mfsd2a exhibited AT2 cell hypertrophy with reduced total surfactant PL levels because of reductions in the most abundant surfactants, PC containing dipalmitic acid, and PC species containing the omega-3 fatty acid docosahexaenoic acid. These changes in surfactant levels and composition were mirrored by similar changes in the AT2 cell lipidome. Mechanistically, direct tracheal instillation of fluorescent LPC and PC probes indicated that Mfsd2a mediates the uptake of LPC generated by pulmonary phospholipase activity in the alveolar space. These studies reveal that Mfsd2a-mediated LPC uptake is quantitatively important in maintaining surfactant homeostasis and identify this lipid transporter as a physiological component of surfactant recycling.

LPGAT1 controls the stearate/palmitate ratio of phosphatidylethanolamine and phosphatidylcholine in sn-1 specific remodeling

Most mammalian phospholipids contain a saturated fatty acid at the sn-1 carbon atom and an unsaturated fatty acid at the sn-2 carbon atom of the glycerol backbone group. While the sn-2 linked chains undergo extensive remodeling by deacylation and reacylation (Lands cycle), it is not known how the composition of saturated fatty acids is controlled at the sn-1 position. Here, we demonstrate that lysophosphatidylglycerol acyltransferase 1 (LPGAT1) is an sn-1 specific acyltransferase that controls the stearate/palmitate ratio of phosphatidylethanolamine (PE) and phosphatidylcholine. Bacterially expressed murine LPGAT1 transferred saturated acyl-CoAs specifically into the sn-1 position of lysophosphatidylethanolamine (LPE) rather than lysophosphatidylglycerol and preferred stearoyl-CoA over palmitoyl-CoA as the substrate. In addition, genetic ablation of LPGAT1 in mice abolished 1-LPE:stearoyl-CoA acyltransferase activity and caused a shift from stearate to palmitate species in PE, dimethyl-PE, and phosphatidylcholine. Lysophosphatidylglycerol acyltransferase 1 KO mice were leaner and had a shorter life span than their littermate controls. Finally, we show that total lipid synthesis was reduced in isolated hepatocytes of LPGAT1 knockout mice. Thus, we conclude that LPGAT1 is an sn-1 specific LPE acyltransferase that controls the stearate/palmitate homeostasis of PE and the metabolites of the PE methylation pathway and that LPGAT1 plays a central role in the regulation of lipid biosynthesis with implications for body fat content and longevity.

Anticancer activity and metabolic profile alterations by ortho-topolin riboside in in vitro and in vivo models of non-small cell lung cancer.

Lung cancer has the highest incidence and mortality rates among all types of cancer worldwide, and 80%-85% of patients with lung cancer are diagnosed with non-small cell lung cancer (NSCLC), which has 5-year survival rate of only 5% at advanced stages. Development of new therapeutic agents and strategies is required to enhance the treatment efficiency in patients with NSCLC. Metabolic alterations and anticancer effects of plant hormones and their derivatives have not been investigated in NSCLC in vitro and in vivo. The present study investigated the cytotoxic effects of 11 plant hormones and their derivatives against NSCLC cell lines; ortho-topolin riboside (oTR) showed the highest cytotoxicity among all tested compounds against NSCLC cells. Alteration of metabolites and lipids was investigated using gas chromatography-mass spectrometry and nano electrospray ionization-mass spectrometry in oTR-treated NSCLC cells and a xenograft mouse model. oTR reduced amino acid and pyrimidine synthesis in NSCLC cells and xenograft tumors. Moreover, oTR reduced glycolytic function and decreased mitochondrial respiration function by inhibiting glutamine and fatty acid oxidation. Increased levels of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine species suggested that oTR might act as a fatty acid oxidation inhibitor. In addition, the increased level of phosphatidylserine species implied that phosphatidylserine-mediated apoptosis occurred in oTR-treated NSCLC cells and xenograft tumor. The antiproliferative and apoptotic effects of oTR were mediated by the reduced p-ERK and p-AKT levels and increased cleaved Caspase-3levels, respectively. This is the first study to investigate the metabolic alterations and anticancer activity of oTR in in vitro and in vivo models of NSCLC. Our results provide basis for the development of oTR-based therapeutic agent for patients with NSCLC.

Screening of Phospholipids in Plasma of Large-Artery Atherosclerotic and Cardioembolic Stroke Patients With Hydrophilic Interaction Chromatography-Mass Spectrometry.

Ischemic stroke (IS) is a deadly and debilitating disease with a high incidence and recurrence rate in elderly people worldwide. Large-artery atherosclerotic (LAA) and cardioembolic (CE) stroke are two leading subtypes and require different management. As a complementary biochemistry method for current diagnostic techniques, a sensitive and accurate phospholipid (PL) targeted lipidomic method was developed in this study. Plasma PLs were selectively extracted with titanium dioxide/fibrous silica nanosphere material, then characterized and quantified with hydrophilic interaction chromatography-mass spectrometry. A total of 31 molecular species of PLs were determined and ten biomarkers including seven molecular species of sphingomyelins (SM d18:1/18:1, d18:1/18:0, d18:1/24:1, d18:1/16:1, d18:1/22:1, d18:1/24:2, and d18:1/16:0) and three molecular species of phosphatidylcholines (16:0/18:1, 16:0/18:2 and 16:0/22:6) showed significant differences in LAA, CE, and healthy control (HC) groups. The independent diagnostic capabilities of these PL biomarkers were successfully evaluated and validated with receiver operating characteristic curves. Additionally, the oleic acid-enriched SMs, which can result in atherogenic lipoprotein aggregation, were proved to be positively related to IS and may perform as the potential risk factors in the future. Meanwhile, valuable suggestions for dietary interventions as an essential source of endogenous PLs could be obtained from this study.

Comprehensive lipidomic analysis of the lipids extracted from freshwater fish bones and crustacean shells.

A comprehensive lipidomic analysis of the lipids extracted from grass carp bones, black carp bones, shrimp shells, and crab shells was performed in this study. First, HPLC analysis revealed that the lipids extracted from shrimp and crab shells contained 60.65% and 77.25% of diacylglycerols, respectively. Second, GC‐MS analysis identified 18 fatty acid species in the lipids extracted from fish bones and crustacean shells, in which polyunsaturated fatty acids (PUFAs) were highly enriched. PUFAs were present at 45.43% in the lipids extracted from shrimp shells. Notably, the lipids extracted from shrimp and crab shells contained a considerable amount of eicosapentaenoic acids and docosahexaenoic acids. Finally, multidimensional mass spectrometry‐based shotgun lipidomics showed that various lipids including acetyl‐L‐carnitine, sphingomyelin (SM), lysophosphatidylcholine, and phosphatidylcholine (PC) were all identified in the lipid samples, but PC and SM were the most abundant. Specifically, the total content of PC in shrimp shells was as high as 6.145 mmol/g. More than 35 species of PC were found in all samples, which were more than other lipids. This study is expected to provide a scientific basis for the application of freshwater fish bones and crustacean shells in food, medicine, and other fields.