Lipidomic profiling of influenza infection identifies mediators that induce and resolve inflammation.

Bioactive lipid mediators play a crucial role in the induction and resolution of inflammation. To elucidate their involvement during influenza infection, liquid chromatography/mass spectrometry lipidomic profiling of 141 lipid species was performed on a mouse influenza model using two viruses of significantly different pathogenicity. Infection by the low-pathogenicity strain X31/H3N2 induced a proinflammatory response followed by a distinct anti-inflammatory response; infection by the high-pathogenicity strain PR8/H1N1 resulted in overlapping pro- and anti-inflammatory states. Integration of the large-scale lipid measurements with targeted gene expression data demonstrated that 5-lipoxygenase metabolites correlated with the pathogenic phase of the infection, whereas 12/15-lipoxygenase metabolites were associated with the resolution phase. Hydroxylated linoleic acid, specifically the ratio of 13- to 9-hydroxyoctadecadienoic acid, was identified as a potential biomarker for immune status during an active infection. Importantly, some of the findings from the animal model were recapitulated in studies of human nasopharyngeal lavages obtained during the 2009-2011 influenza seasons.

Lipidomics of phospholipase A2 reveals exquisite specificity in macrophages.

Phospholipase A2 (PLA2) constitutes a superfamily of enzymes that hydrolyze phospholipids at their sn-2 fatty acyl position. Our laboratory has demonstrated that PLA2 enzymes regulate membrane remodeling and cell signaling by their specificity toward their phospholipid substrates at the molecular level. Recent in vitro studies show that each type of PLA2, including Group IVA cytosolic PLA2 (cPLA2), Group V secreted PLA2 (sPLA2), Group VIA calcium independent PLA2 (iPLA2) and Group VIIA lipoprotein-associated PLA2, also known as platelet-activating factor acetyl hydrolase, can discriminate exquisitely between fatty acids at the sn-2 position. Thus, these enzymes regulate the production of diverse PUFA precursors of inflammatory metabolites. We now determined PLA2 specificity in macrophage cells grown in cell culture, where the amounts and localization of the phospholipid substrates play a role in which specific phospholipids are hydrolyzed by each enzyme type. We used PLA2 stereospecific inhibitors in tandem with a novel UPLC-MS/MS-based lipidomics platform to quantify more than a thousand unique phospholipid molecular species demonstrating cPLA2, sPLA2, and iPLA2 activity and specificity toward the phospholipids in living cells. The observed specificity follows the in vitro capability of the enzymes and can reflect the enrichment of certain phospholipid species in specific membrane locations where particular PLA2's associate. For assaying, we target 20:4-PI for cPLA2, 22:6-PG for sPLA2, and 18:2-PC for iPLA2. These new results provide great insight into the physiological role of PLA2 enzymes in cell membrane remodeling and could shed light on how PLA2 enzymes underpin inflammation and other lipid-related diseases.

Lipid storage disease in 4 sibling superb birds-of-paradise (Lophorina superba).

Pedigree analysis, clinical, gross, microscopic, ultrastructural, and lipidomic findings in 4 female superb bird-of-paradise (SBOP, Lophorina superba) siblings led to the diagnosis of a primary inherited glycerolipid storage disease. These birds were the offspring of a related breeding pair (inbreeding coefficient = 0.1797) and are the only known SBOPs to display this constellation of lesions. The birds ranged from 0.75 to 4.3 years of age at the time of death. Two birds were euthanized and 1 died naturally due to the disease, and 1 died of head trauma with no prior clinical signs. Macroscopic findings included hepatomegaly and pallor (4/4), cardiac and renal pallor (2/4), and coelomic effusion (1/4). Microscopic examination found marked tissue distortion due to cytoplasmic lipid vacuoles in hepatocytes (4/4), cardiomyocytes (4/4), renal tubular epithelial cells (4/4), parathyroid gland principal cells (2/2), exocrine pancreatic cells (3/3), and the glandular cells of the ventriculus and proventriculus (3/3). Ultrastructurally, the lipids were deposited in single to coalescing or fused droplets lined by an inconspicuous or discontinuous monolayer membrane. Lipidomic profiling found that the cytoplasmic lipid deposits were primarily composed of triacylglycerols. Future work, including sequencing of the SBOP genome and genotyping, will be required to definitively determine the underlying genetic mechanism of this disease.

Influence of Dietary n-3 Long Chain Polyunsaturated Fatty Acid Intake on Oxylipins in Erythrocytes of Women with Rheumatoid Arthritis.

Oxylipins derived from n-3 fatty acids are suggested as the link between these fatty acids and reduced inflammation. The aim of the present study was to explore the effect of a randomized controlled cross-over intervention on oxylipin patterns in erythrocytes. Twenty-three women with rheumatoid arthritis completed 2 × 11-weeks exchanging one cooked meal per day, 5 days a week, for a meal including 75 g blue mussels (source for n-3 fatty acids) or 75 g meat. Erythrocyte oxylipins were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results were analyzed with multivariate data analysis. Orthogonal projections to latent structures (OPLS) with effect projections and with discriminant analysis were performed to compare the two diets' effects on oxylipins. Wilcoxon signed rank test was used to test pre and post values for each dietary period as well as post blue-mussel vs. post meat. The blue-mussel diet led to significant changes in a few oxylipins from the precursor fatty acids arachidonic acid and dihomo-É£-linolenic acid. Despite significant changes in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and free EPA in erythrocytes in the mussel group, no concurrent changes in their oxylipins were seen. Further research is needed to study the link between n-3 fatty-acid intake, blood oxylipins, and inflammation.

Automated Annotation of Sphingolipids Including Accurate Identification of Hydroxylation Sites Using MSn Data.

Sphingolipids constitute a heterogeneous lipid category that is involved in many key cellular functions. For high-throughput analyses of sphingolipids, tandem mass spectrometry (MS/MS) is the method of choice, offering sufficient sensitivity, structural information, and quantitative precision for detecting hundreds to thousands of species simultaneously. While glycerolipids and phospholipids are predominantly non-hydroxylated, sphingolipids are typically dihydroxylated. However, species containing one or three hydroxylation sites can be detected frequently. This variability in the number of hydroxylation sites on the sphingolipid long-chain base and the fatty acyl moiety produces many more isobaric species and fragments than for other lipid categories. Due to this complexity, the automated annotation of sphingolipid species is challenging, and incorrect annotations are common. In this study, we present an extension of the Lipid Data Analyzer (LDA) "decision rule set" concept that considers the structural characteristics that are specific for this lipid category. To address the challenges inherent to automated annotation of sphingolipid structures from MS/MS data, we first developed decision rule sets using spectra from authentic standards and then tested the applicability on biological samples including murine brain and human plasma. A benchmark test based on the murine brain samples revealed a highly improved annotation quality as measured by sensitivity and reliability. The results of this benchmark test combined with the easy extensibility of the software to other (sphingo)lipid classes and the capability to detect and correctly annotate novel sphingolipid species make LDA broadly applicable to automated sphingolipid analysis, especially in high-throughput settings.

Deciphering lipid structures based on platform-independent decision rules.

We achieve automated and reliable annotation of lipid species and their molecular structures in high-throughput data from chromatography-coupled tandem mass spectrometry using decision rule sets embedded in Lipid Data Analyzer (LDA; http://genome.tugraz.at/lda2). Using various low- and high-resolution mass spectrometry instruments with several collision energies, we proved the method's platform independence. We propose that the software's reliability, flexibility, and ability to identify novel lipid molecular species may now render current state-of-the-art lipid libraries obsolete.

AMP-activated protein kinase activation ameliorates eicosanoid dysregulation in high-fat-induced kidney disease in mice.

High-fat diet (HFD) causes renal lipotoxicity that is ameliorated with AMP-activated protein kinase (AMPK) activation. Although bioactive eicosanoids increase with HFD and are essential in regulation of renal disease, their role in the inflammatory response to HFD-induced kidney disease and their modulation by AMPK activation remain unexplored. In a mouse model, we explored the effects of HFD on eicosanoid synthesis and the role of AMPK activation in ameliorating these changes. We used targeted lipidomic profiling with quantitative MS to determine PUFA and eicosanoid content in kidneys, urine, and renal arterial and venous circulation. HFD increased phospholipase expression as well as the total and free pro-inflammatory arachidonic acid (AA) and anti-inflammatory DHA in kidneys. Consistent with the parent PUFA levels, the AA- and DHA-derived lipoxygenase (LOX), cytochrome P450, and nonenzymatic degradation (NE) metabolites increased in kidneys with HFD, while EPA-derived LOX and NE metabolites decreased. Conversely, treatment with 5-aminoimidazole-4-carboxamide-1-ß-D-furanosyl 5'-monophosphate (AICAR), an AMPK activator, reduced the free AA and DHA content and the DHA-derived metabolites in kidney. Interestingly, kidney and circulating AA, AA metabolites, EPA-derived LOX, and NE metabolites are increased with HFD; whereas, DHA metabolites are increased in kidney in contrast to their decreased circulating levels with HFD. Together, these changes showcase HFD-induced pro- and anti-inflammatory eicosanoid dysregulation and highlight the role of AMPK in correcting HFD-induced dysregulated eicosanoid pathways.

Pro- and anti-inflammatory eicosanoids in psoriatic arthritis.

INTRODUCTION: Eicosanoids are biological lipids that serve as both activators and suppressors of inflammation. Eicosanoid pathways are implicated in synovitis and joint destruction in inflammatory arthritis, yet they might also have a protective function, underscoring the need for a comprehensive understanding of how eicosanoid pathways might be imbalanced. Until recently, sensitive and scalable methods for detecting and quantifying a high number of eicosanoids have not been available. OBJECTIVE: Here, we intend to describe a detailed eicosanoid profiling in patients with psoriatic arthritis (PsA) and evaluate correlations with parameters of disease activity. METHODS: Forty-one patients with PsA, all of whom satisfied the CASPAR classification criteria for PsA, were studied. Outcomes reflecting the activity of peripheral arthritis as well as skin psoriasis, Disease Activity Score (DAS)28, Clinical Disease Index (CDAI) and Body Surface Area (BSA) were assessed. Serum eicosanoids were determined by LC-MS, and the correlation between metabolite levels and disease scores was evaluated. RESULTS: Sixty-six eicosanoids were identified by reverse-phase LC/MS. Certain eicosanoids species including several pro-inflammatory eicosanoids such as PGE2, HXB3 or 6,15-dk,dh,PGF1a correlated with joint disease score. Several eicosapentaenoic acid (EPA)-derived eicosanoids, which associate with anti-inflammatory properties, such as 11-HEPE, 12-HEPE and 15-HEPE, correlated with DAS28 (Disease Activity Score) and CDAI (Clinical Disease Activity Index) as well. Of interest, resolvin D1, a DHA-derived anti-inflammatory eicosanoid, was down-regulated in patients with high disease activity. CONCLUSION: Both pro- and anti-inflammatory eicosanoids were associated with joint disease score, potentially representing pathways of harm as well as benefit. Further studies are needed to determine whether these eicosanoid species might also play a role in the pathogenesis of joint inflammation in PsA.

Quantitative determination of esterified eicosanoids and related oxygenated metabolites after base hydrolysis.

Eicosanoids and related metabolites (oxylipins) possess potent signaling properties, elicit numerous important physiologic responses, and serve as biomarkers of disease. In addition to their presence in free form, a considerable portion of these bioactive lipids is esterified to complex lipids in cell membranes and plasma lipoproteins. We developed a rapid and sensitive method for the analysis of esterified oxylipins using alkaline hydrolysis to release them followed by ultra-performance LC coupled with mass spectrometric analysis. Detailed evaluation of the data revealed that several oxylipins are susceptible to alkaline-induced degradation. Nevertheless, of the 136 metabolites we examined, 56 were reproducibly recovered after alkaline hydrolysis. We classified those metabolites that were resistant to alkaline-induced degradation and applied this methodology to quantify metabolite levels in a macrophage cell model and in plasma of healthy subjects. After alkaline hydrolysis of lipids, 34 metabolites could be detected and quantified in resting and activated macrophages, and 38 metabolites were recovered from human plasma at levels that were substantially greater than in free form. By carefully selecting internal standards and taking the observed experimental limitations into account, we established a robust method that can be reliably employed for the measurement of esterified oxylipins in biological samples.

Failure to up-regulate transcription of genes necessary for muscle adaptation underlies limb girdle muscular dystrophy 2A (calpainopathy).

Limb girdle muscular dystrophy 2A is due to loss-of-function mutations in the Calpain 3 (CAPN3) gene. Our previous data suggest that CAPN3 helps to maintain the integrity of the triad complex in skeletal muscle. In Capn3 knock-out mice (C3KO), Ca2+ release and Ca2+/calmodulin kinase II (CaMKII) signaling are attenuated. We hypothesized that calpainopathy may result from a failure to transmit loading-induced Ca2+-mediated signals, necessary to up-regulate expression of muscle adaptation genes. To test this hypothesis, we compared transcriptomes of muscles from wild type (WT) and C3KO mice subjected to endurance exercise. In WT mice, exercise induces a gene signature that includes myofibrillar, mitochondrial and oxidative lipid metabolism genes, necessary for muscle adaptation. C3KO muscles fail to activate the same gene signature. Furthermore, in agreement with the aberrant transcriptional profile, we observe a commensurate functional defect in lipid metabolism whereby C3KO muscles fail to release fatty acids from stored triacylglycerol. In conjunction with the defects in oxidative metabolism, C3KO mice demonstrate reduced exercise endurance. Failure to up-regulate genes in C3KO muscles is due, in part, to decreased levels of PGC1α, a transcriptional co-regulator that orchestrates the muscle adaptation response. Destabilization of PGC1α is attributable to decreased p38 MAPK activation via diminished CaMKII signaling. Thus, we elucidate a pathway downstream of Ca2+-mediated CaMKII activation that is dysfunctional in C3KO mice, leading to reduced transcription of genes involved in muscle adaptation. These studies identify a novel mechanism of muscular dystrophy: a blunted transcriptional response to muscle loading resulting in chronic failure to adapt and remodel.

Harmonizing lipidomics: NIST interlaboratory comparison exercise for lipidomics using SRM 1950-Metabolites in Frozen Human Plasma.

As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950-Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement.

Biomarkers of NAFLD progression: a lipidomics approach to an epidemic.

The spectrum of nonalcoholic fatty liver disease (NAFLD) includes steatosis, nonalcoholic steatohepatitis (NASH), and cirrhosis. Recognition and timely diagnosis of these different stages, particularly NASH, is important for both potential reversibility and limitation of complications. Liver biopsy remains the clinical standard for definitive diagnosis. Diagnostic tools minimizing the need for invasive procedures or that add information to histologic data are important in novel management strategies for the growing epidemic of NAFLD. We describe an "omics" approach to detecting a reproducible signature of lipid metabolites, aqueous intracellular metabolites, SNPs, and mRNA transcripts in a double-blinded study of patients with different stages of NAFLD that involves profiling liver biopsies, plasma, and urine samples. Using linear discriminant analysis, a panel of 20 plasma metabolites that includes glycerophospholipids, sphingolipids, sterols, and various aqueous small molecular weight components involved in cellular metabolic pathways, can be used to differentiate between NASH and steatosis. This identification of differential biomolecular signatures has the potential to improve clinical diagnosis and facilitate therapeutic intervention of NAFLD.

Three-dimensional enhanced lipidomics analysis combining UPLC, differential ion mobility spectrometry, and mass spectrometric separation strategies.

Phospholipids serve as central structural components in cellular membranes and as potent mediators in numerous signaling pathways. There are six main classes of naturally occurring phospholipids distinguished by their distinct polar head groups that contain many unique molecular species with distinct fatty acid composition. Phospholipid molecular species are often expressed as isobaric species that are denoted by the phospholipid class and the total number of carbon atoms and double bonds contained in the esterified fatty acyl groups (e.g., phosphatidylcholine 34:2). Techniques to separate these molecules exist, and each has positive and negative attributes. Hydrophilic interaction liquid chromatography uses polar bonded silica to separate lipids by polar head group but not by specific molecular species. Reversed phase (RP) chromatography can separate by fatty acyl chain composition but not by polar head group. Herein we describe a new strategy called differential ion mobility spectrometry (DMS), which separates phospholipid classes by their polar head group. Combining DMS with current LC methods enhances phospholipid separation by increasing resolution, specificity, and signal-to-noise ratio. Additional application of specialized information-dependent acquisition methodologies along with RP chromatography allows full isobaric resolution, identification, and compositional characterization of specific phospholipids at the molecular level.

Targeted proteomics of the eicosanoid biosynthetic pathway completes an integrated genomics-proteomics-metabolomics picture of cellular metabolism.

Eicosanoids constitute a diverse class of bioactive lipid mediators that are produced from arachidonic acid and play critical roles in cell signaling and inflammatory aspects of numerous diseases. We have previously quantified eicosanoid metabolite production in RAW264.7 macrophage cells in response to Toll-like receptor 4 signaling and analyzed the levels of transcripts coding for the enzymes involved in the eicosanoid metabolite biosynthetic pathways. We now report the quantification of changes in protein levels under similar experimental conditions in RAW264.7 macrophages by multiple reaction monitoring mass spectrometry, an accurate targeted protein quantification method. The data complete the first fully integrated genomic, proteomic, and metabolomic analysis of the eicosanoid biochemical pathway.

Homeocurvature adaptation of phospholipids to pressure in deep-sea invertebrates.

Hydrostatic pressure increases with depth in the ocean, but little is known about the molecular bases of biological pressure tolerance. We describe a mode of pressure adaptation in comb jellies (ctenophores) that also constrains these animals' depth range. Structural analysis of deep-sea ctenophore lipids shows that they form a nonbilayer phase at pressures under which the phase is not typically stable. Lipidomics and all-atom simulations identified phospholipids with strong negative spontaneous curvature, including plasmalogens, as a hallmark of deep-adapted membranes that causes this phase behavior. Synthesis of plasmalogens enhanced pressure tolerance in Escherichia coli, whereas low-curvature lipids had the opposite effect. Imaging of ctenophore tissues indicated that the disintegration of deep-sea animals when decompressed could be driven by a phase transition in their phospholipid membranes.

High sensitivity quantitative lipidomics analysis of fatty acids in biological samples by gas chromatography-mass spectrometry.

Historically considered to be simple membrane components serving as structural elements and energy storing entities, fatty acids are now increasingly recognized as potent signaling molecules involved in many metabolic processes. Quantitative determination of fatty acids and exploration of fatty acid profiles have become common place in lipid analysis. We present here a reliable and sensitive method for comprehensive analysis of free fatty acids and fatty acid composition of complex lipids in biological material. The separation and quantitation of fatty acids are achieved by capillary gas chromatography. The analytical method uses pentafluorobenzyl bromide derivatization and negative chemical ionization gas chromatography-mass spectrometry. The chromatographic procedure provides base line separation between saturated and unsaturated fatty acids of different chain lengths as well as between most positional isomers. Fatty acids are extracted in the presence of isotope-labeled internal standards for high quantitation accuracy. Mass spectrometer conditions are optimized for broad detection capacity and sensitivity capable of measuring trace amounts of fatty acids in complex biological samples. .

Human tau mutations in cerebral organoids induce a progressive dyshomeostasis of cholesterol.

Mutations in the MAPT gene that encodes tau lead to frontotemporal dementia (FTD) with pathology evident in both cerebral neurons and glia. Human cerebral organoids (hCOs) from individuals harboring pathogenic tau mutations can reveal the earliest downstream effects on molecular pathways within a developmental context, generating interacting neurons and glia. We found that in hCOs carrying the V337M and R406W tau mutations, the cholesterol biosynthesis pathway in astrocytes was the top upregulated gene set compared with isogenic controls by single-cell RNA sequencing (scRNA-seq). The 15 upregulated genes included HMGCR, ACAT2, STARD4, LDLR, and SREBF2. This result was confirmed in a homozygous R406W mutant cell line by immunostaining and sterol measurements. Cholesterol abundance in the brain is tightly regulated by efflux and cholesterol biosynthetic enzyme levels in astrocytes, and dysregulation can cause aberrant phosphorylation of tau. Our findings suggest that cholesterol dyshomeostasis is an early event in the etiology of neurodegeneration caused by tau mutations.

De novo sphingolipid biosynthesis necessitates detoxification in cancer cells.

Sphingolipids play important signaling and structural roles in cells. Here, we find that during de novo sphingolipid biosynthesis, a toxic metabolite is formed with critical implications for cancer cell survival. The enzyme catalyzing the first step in this pathway, serine palmitoyltransferase complex (SPT), is upregulated in breast and other cancers. SPT is dispensable for cancer cell proliferation, as sphingolipids can be salvaged from the environment. However, SPT activity introduces a liability as its product, 3-ketodihydrosphingosine (3KDS), is toxic and requires clearance via the downstream enzyme 3-ketodihydrosphingosine reductase (KDSR). In cancer cells, but not normal cells, targeting KDSR induces toxic 3KDS accumulation leading to endoplasmic reticulum (ER) dysfunction and loss of proteostasis. Furthermore, the antitumor effect of KDSR disruption can be enhanced by increasing metabolic input (via high-fat diet) to allow greater 3KDS production. Thus, de novo sphingolipid biosynthesis entails a detoxification requirement in cancer cells that can be therapeutically exploited.

Effect of gestational hypercholesterolemia and maternal immunization on offspring plasma eicosanoids.

OBJECTIVE: Maternal immunization with oxidized low-density lipoprotein prior to pregnancy prevents pathogenic in utero programming by gestational hypercholesterolemia, but it is unknown whether gestational hypercholesterolemia and maternal immunization affect similar pathways. STUDY DESIGN: A lipidomic approach was used for unbiased plasma eicosanoid profiling in adult offspring of immunized and nonimmunized normocholesterolemic or hypercholesterolemic rabbit mothers. RESULTS: Gestational hypercholesterolemia was associated with increased levels of some eicosanoids formed by the cyclooxygenase and 12-lipoxygenase pathways only (including thromboxane B2, prostaglandin [PG] F2α, PGE2, and PGD2). Immunization of hypercholesterolemic or normocholesterolemic mothers reduced 9 of 14 eicosanoids of the cyclooxygenase pathway, 21 of 23 eicosanoids of the 5- and 12-lipoxygenase pathways (eg, 5-hydroxyeicosatetraenoic acid, hepoxilin B3, 12-hydroxyeicosatetraenoic acid), 8 of 19 eicosanoids of the cytochrome P-450 pathway, and all metabolites of the nonenzymatic pathway. CONCLUSION: Maternal immunization not only counteracts in utero programming by gestational hypercholesterolemia but reduces a broad range of eicosanoid modulators of immunity and inflammation in offspring.