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The increasing focus on lipid metabolism has revealed a need for analytical techniques capable of structurally characterizing lipids with a high degree of specificity. Lipids can exist as any one of a large number of double bond positional isomers, which are indistinguishable by single-stage mass spectrometry alone. Ozonolysis reactions coupled to mass spectrometry have previously been demonstrated as a means for localizing double bonds in unsaturated lipids. Here we describe an online, solution-phase reactor using ozone produced via a low-pressure mercury lamp, which generates aldehyde products diagnostic of cleavage at a particular double bond position. This flow-cell device is utilized in conjunction with structurally selective ion mobility-mass spectrometry. The lamp-mediated reaction was found to be effective for multiple lipid species in both positive and negative ionization modes, and the conversion efficiency from precursor to product ions was tunable across a wide range (20-95%) by varying the flow rate through the ozonolysis device. Ion mobility separation of the ozonolysis products generated additional structural information and revealed the presence of saturated species in a complex mixture. The method presented here is simple, robust, and readily coupled to existing instrument platforms with minimal modifications necessary. For these reasons, application to standard lipidomic workflows is possible and aids in more comprehensive structural characterization of a myriad of lipid species.
Stargardt disease is a juvenile onset retinal degeneration, associated with elevated levels of lipofuscin and its bis-retinoid components, such as N-retinylidene-N-retinylethanolamine (A2E). However, the pathogenesis of Stargardt is still poorly understood and targeted treatments are not available. Utilizing high spatial and high mass resolution matrix assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS), we determined alterations of lipid profiles specifically localized to the retinal pigment epithelium (RPE) in Abca4 Stargardt model mice compared to their relevant background strain. Extensive analysis by LC-MS/MS in both positive and negative ion mode was required to accurately confirm the identity of one highly expressed lipid class, bis(monoacylgylercoro)phosphate (BMP) lipids, and to distinguish them from isobaric species. The same BMP lipids were also detected in the RPE of healthy human retina. BMP lipids have been previously associated with the endosomal/lysosomal storage diseases Niemann-Pick and neuronal ceroid lipofuscinosis and have been reported to regulate cholesterol levels in endosomes. These results suggest that perturbations in lipid metabolism associated with late endosomal/lysosomal dysfunction may play a role in the pathogenesis of Stargardt disease and is evidenced in human retinas.
Standard harvest and preparation of human saphenous vein (HSV) for autologous coronary and peripheral arterial bypass procedures is associated with injury and increased oxidative stress that negatively affect graft performance. In this study we investigated the global metabolomic profiles of HSV before (unprepared; UP) and after standard vein graft preparation (AP). AP-HSV showed impaired vasomotor function that was associated with increased oxidative stress, phospholipid hydrolysis and energy depletion that are characteristic of mechanical and chemical injury. A porcine model (PSV) was utilized to validate these metabolomic changes in HSV and to determine the efficacy of an improved preparation technique (OP) using pressure-regulated distension, a non-toxic vein marker, and graft storage in buffered PlasmaLyte solution in limiting metabolic decompensation due to graft preparation. Deficits in vasomotor function and metabolic signature observed in AP-PSV could be largely mitigated with the OP procedure. These findings suggest that simple strategies aimed at reducing injury during graft harvest and preparation represents a straightforward and viable strategy to preserve conduit function and possibly improve graft patency.
Huntington's disease is characterized by a complex and heterogeneous pathogenic profile. Studies have shown that disturbance in lipid homeostasis may represent a critical determinant in the progression of several neurodegenerative disorders. The recognition of perturbed lipid metabolism is only recently becoming evident in HD. In order to provide more insight into the nature of such a perturbation and into the effect its modulation may have in HD pathology, we investigated the metabolism of Sphingosine-1-phosphate (S1P), one of the most important bioactive lipids, in both animal models and patient samples. Here, we demonstrated that S1P metabolism is significantly disrupted in HD even at early stage of the disease and importantly, we revealed that such a dysfunction represents a common denominator among multiple disease models ranging from cells to humans through mouse models. Interestingly, the in vitro anti-apoptotic and the pro-survival actions seen after modulation of S1P-metabolizing enzymes allows this axis to emerge as a new druggable target and unfolds its promising therapeutic potential for the development of more effective and targeted interventions against this incurable condition.
Dietary intake of PUFA has been associated with colorectal neoplasm risk; however, results from observational studies have been inconsistent. Most prior studies have utilised self-reported dietary measures to assess fatty acid exposure which might be more susceptible to measurement error and biases compared with biomarkers. The purpose of this study was to determine whether erythrocyte phospholipid membrane PUFA percentages are associated with colorectal adenoma risk. We included data from 904 adenoma cases and 835 polyp-free controls who participated in the Tennessee Colorectal Polyp Study, a large colonoscopy-based case-control study. Erythrocyte membrane PUFA percentages were measured using GC. Conditional logistic regression was used to calculate adjusted OR for risk of colorectal adenomas with erythrocyte membrane PUFA. Higher erythrocyte membrane percentages of arachidonic acid was associated with an increased risk of colorectal adenomas (adjusted OR 1·66; 95 % CI 1·05, 2·62, P trend=0·02) comparing the highest tertile to the lowest tertile. The effect size for arachidonic acid was more pronounced when restricting the analysis to advanced adenomas only. Higher erythrocyte membrane EPA percentages were associated with a trend towards a reduced risk of advanced colorectal adenomas (P trend=0·05). Erythrocyte membrane arachidonic acid percentages are associated with an increased risk of colorectal adenomas.
Hibernating mammals, like the arctic ground squirrel (AGS), exhibit robust resistance to myocardial ischemia/reperfusion (IR) injury. Regulated preference for lipid over glucose to fuel metabolism may play an important role. We tested whether providing lipid in an emulsion protects hearts from summer-active AGS better than hearts from Brown Norway (BN) rats against normothermic IR injury. Langendorff-prepared AGS and BN rat hearts were perfused with Krebs solution containing 7.5 mM glucose with or without 1% Intralipid™. After stabilization and cardioplegia, hearts underwent 45-min global ischemia and 60-min reperfusion. Coronary flow, isovolumetric left ventricular pressure, and mitochondrial redox state were measured continuously; infarct size was measured at the end of the experiment. Glucose-only AGS hearts functioned significantly better on reperfusion than BN rat hearts. Intralipid™ administration resulted in additional functional improvement in AGS compared to glucose-only and BN rat hearts. Infarct size was not different among groups. Even under non-hibernating conditions, AGS hearts performed better after IR than the best-protected rat strain. This, however, appears to strongly depend on metabolic fuel: Intralipid™ led to a significant improvement in return of function in AGS, but not in BN rat hearts, suggesting that year-round endogenous mechanisms are involved in myocardial lipid utilization that contributes to improved cardiac performance, independent of the metabolic rate decrease during hibernation. Comparative lipid analysis revealed four candidates as possible cardioprotective lipid groups. The improved function in Intralipid™-perfused AGS hearts also challenges the current paradigm that increased glucose and decreased lipid metabolism are favorable during myocardial IR.
Oxidative stress and inflammation are two major contributing factors to atherosclerosis, a leading cause of cardiovascular disease. Oxidation of phospholipids on the surface of low density lipoprotein (LDL) particles generated under oxidative stress has been associated with the progression of atherosclerosis, but the underlying molecular mechanisms remain poorly defined. We identified a novel series of oxidation products containing the cyclopentenone moiety, termed deoxy-A/J-isoprostanes-phosphocholine, from 1-palmitoyl-2-arachidonoyl--glycero-3-phosphocholine using mass spectrometry and by comparison to a chemically synthesized standard. Transcriptomic analysis (RNA-seq) demonstrated that these compounds affected >200 genes in bone marrow-derived macrophages, and genes associated with inflammatory and anti-oxidative responses are among the top 5 differentially expressed. To further investigate the biological relevance of these novel oxidized phospholipids in atherosclerosis, we chemically synthesized a representative compound 1-palmitoyl-2-15-deoxy-δ-12,14-prostaglandin J--glycero-3-phosphocholine (15d-PGJ-PC) and found that it induced anti-inflammatory and anti-oxidant responses in macrophages through modulation of NF-κB, peroxisome proliferator-activated receptor γ (PPARγ), and Nrf2 pathways; this compound also showed potent anti-inflammatory properties in a mice model of LPS-induced systematic inflammatory response syndrome. Additionally, 15d-PGJ-PC inhibited macrophage foam cell formation, suggesting a beneficial role against atherosclerosis. These properties were consistent with decreased levels of these compounds in the plasma of patients with coronary heart disease compared with control subjects. Our findings uncovered a novel molecular mechanism for the negative regulation of inflammation and positive enhancement of anti-oxidative responses in macrophages by these oxidized phospholipids in LDL in the context of atherosclerosis.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
The cellular production of free radicals or reactive oxygen species (ROS) can lead to protein, lipid or DNA modifications and tumor formation. The cellular lipids undergo structural changes through the actions of enzymes (e.g. cyclooxygenases) or free radicals to form a class of compounds called Isolevuglandins (IsoLGs). The recruitment and continued exposure of tissue to ROS and IsoLGs causes increased cell proliferation, mutagenesis, loss of normal cell function and angiogenesis. The elevated concentration of ROS in cancerous tissues suggests that these mediators play an important role in cancer development. We hypothesized that tumors with elevated ROS levels would similarly possess an increased concentration of IsoLGs when compared with normal tissue. Using D11, an ScFv recombinant antibody specific for IsoLGs, we utilized immunohistochemistry to visualize the presence of IsoLG in human tumors compared to normal adjacent tissue (NAT) to the same tumor. We found that IsoLG concentrations were elevated in human breast, colon, kidney, liver, lung, pancreatic and tongue tumor cells when compared to NAT and believe that IsoLGs can be used as a gauge indicative of lipid peroxidation in tumors.
Copyright © 2017 Elsevier Inc. All rights reserved.
Nuclear receptors are ligand-activated transcription factors whose diverse biological functions are classically regulated by cholesterol-based small molecules. Over the past few decades, a growing body of evidence has demonstrated that phospholipids and other similar amphipathic molecules can also specifically bind and functionally regulate the activity of certain nuclear receptors, suggesting a critical role for these non-cholesterol-based molecules in transcriptional regulation. Phosphatidylcholines, phosphoinositides and sphingolipids are a few of the many phospholipid like molecules shown to quite specifically regulate nuclear receptors in mouse models, cell lines and in vitro. More recent evidence has also shown that certain nuclear receptors can "present" a bound phospholipid headgroup to key lipid signaling enzymes, which can then modify the phospholipid headgroup with very unique kinetic properties. Here, we review the broad array of phospholipid/nuclear receptor interactions, from the perspective of the chemical nature of the phospholipid, and the cellular abundance of the phospholipid. We also view the data in the light of well established paradigms for phospholipid mediated transcriptional regulation, as well as newer models of how phospholipids might effect transcription in the acute regulation of complex nuclear signaling pathways. Thus, this review provides novel insight into the new, non-membrane associated roles nuclear phospholipids play in regulating complex nuclear events, centered on the nuclear receptor superfamily of transcription factors.
Copyright © 2016 Elsevier Ltd. All rights reserved.