Systems level analysis of lipidome

Lipids, once thought to be mainly for energy-storage and structural purpose, have now gained immense recognition as a class of critical metabolites with versatile functions. The diversity and complexity of the cellular lipids are the main challenge for the comprehensive analysis of a lipidome. Lipidomics, which aims at mapping all of the lipids in a cell, is expanded rapidly in recent years, mainly attributed to recent advances in mass spectrometry (MS). MS-based lipidomic approaches developed recently allow the quick profiling of hundreds of lipids in a crude lipid extract. With the aid of latest computational tools/software (chemometrics), aberrant lipid metabolites or important signaling lipid(s) could be easily identified using unbiased lipid profiling approaches. Further tandem MS (MS/MS)-based lipidomic approaches, known as targeted approaches and able to convey structural information, hold the promise for high-throughput lipidome analysis. In this review, I discussed the basic strategy for systems level analysis of lipidome in biomedical study.     

Statin action favors normalization of the plasma lipidome in the atherogenic mixed dyslipidemia of MetS: potential relevance to statin-associated dysglycemia

The impact of statin treatment on the abnormal plasma lipidome of mixed dyslipidemic patients with metabolic syndrome (MetS), a group at increased risk of developing diabetes, was evaluated. Insulin-resistant hypertriglyceridemic hypertensive obese males (n = 12) displaying MetS were treated with pitavastatin (4 mg/day) for 180 days; healthy normolipidemic age-matched nonobese males (n = 12) acted as controls. Statin treatment substantially normalized triglyceride (−41%), remnant cholesterol (−55%), and LDL-cholesterol (−39%), with minor effect on HDL-cholesterol (+4%). Lipidomic analysis, normalized to nonHDL-cholesterol in order to probe statin-induced differences in molecular composition independently of reduction in plasma cholesterol, revealed increment in 132 of 138 lipid species that were subnormal at baseline and significantly shifted toward the control group on statin treatment. Increment in alkyl- and alkenylphospholipids (plasmalogens) was prominent, and consistent with significant statin-induced increase in plasma polyunsaturated fatty acid levels. Comparison of the statin-mediated lipidomic changes in MetS with the abnormal plasma lipidomic profile characteristic of prediabetes and T2D in the Australian Diabetes, Obesity, and Lifestyle Study and San Antonio Family Heart Study cohorts by hypergeometric analysis revealed a significant shift toward the lipid profile of controls, indicative of a marked trend toward a normolipidemic phenotype. Pitavastatin attenuated the abnormal plasma lipidome of MetS patients typical of prediabetes and T2D.     

Heterogeneity of lipidomic profiles among lung cancer subtypes of patients

Lung cancer is a leading cause of cancer‐related deaths with an increasing incidence and poor prognoses. To further understand the regulatory mechanisms of lipidomic profiles in lung cancer subtypes, we measure the profiles of plasma lipidome between health and patients with lung cancer or among patients with squamous cell carcinomas, adenocarcinoma or small cell lung cancer and to correct lipidomic and genomic profiles of lipid‐associated enzymes and proteins by integrating the data of large‐scale genome screening. Our studies demonstrated that circulating levels of PS and lysoPS significantly increased, while lysoPE and PE decreased in patients with lung cancer. Our data indicate that lung cancer‐specific and subtype‐specific lipidomics in the circulation are important to understand mechanisms of systemic metabolisms and identify diagnostic biomarkers and therapeutic targets. The carbon atoms, dual bonds or isomerism in the lipid molecule may play important roles in lung cancer cell differentiations and development. This is the first try to integrate lipidomic data with lipid protein‐associated genomic expression among lung cancer subtypes as the part of clinical trans‐omics. We found that a large number of lipid protein‐associated genes significantly change among cancer subtypes, with correlations with altered species and spatial structures of lipid metabolites.     

Lipidomic analysis of the retina in a rat model of Smith-Lemli-Opitz syndrome: alterations in docosahexaenoic acid content of phospholipid molecular species

Smith–Lemli–Opitz syndrome (SLOS) is a complex hereditary disease caused by an enzymatic defect in the last step of cholesterol biosynthesis. Progressive retinal degeneration occurs in an AY9944-induced rat model of SLOS, with biochemical and electroretinographic hallmarks comparable with the human disease. We evaluated alterations in the non-sterol lipid components of the retina in this model, compared with age-matched controls, using lipidomic analysis. The levels of 16:0–22:6 and 18:0–22:6 phosphatidylcholine molecular species in retinas were less by > 50% and > 33%, respectively, in rats treated for either 2 or 3 months with AY9944. Relative to controls, AY9944 treatment resulted in > 60% less di-22:6 and > 15% less 18:0–22:6 phosphatidylethanolamine molecular species. The predominant phosphatidylserine (PS) molecular species in control retinas were 18:0–22:6 and di-22:6; notably, AY9944 treatment resulted in > 80% less di-22:6 PS, relative to controls. Remarkably, these changes occurred in the absence of n3 fatty acid deficiency in plasma or liver. Thus, the retinal lipidome is globally altered in the SLOS rat model, relative to control rats, with the most profound changes being less phosphatidylcholine, phosphatidylethanolamine, and PS molecular species containing docosahexaenoic acid (22:6). These findings suggest that SLOS may involve additional metabolic compromise beyond the primary enzymatic defect in the cholesterol pathway.     

Validation of a multiplexed and targeted lipidomics assay for accurate quantification of lipidomes

A major challenge of lipidomics is to determine and quantify the precise content of complex lipidomes to the exact lipid molecular species. Often, multiple methods are needed to achieve sufficient lipidomic coverage to make these determinations. Multiplexed targeted assays offer a practical alternative to enable quantitative lipidomics amenable to quality control standards within a scalable platform. Herein, we developed a multiplexed normal phase liquid chromatography-hydrophilic interaction chromatography multiple reaction monitoring method that quantifies lipid molecular species across over 20 lipid classes spanning wide polarities in a single 20-min run. Analytical challenges such as in-source fragmentation, isomer separations, and concentration dynamics were addressed to ensure confidence in selectivity, quantification, and reproducibility. Utilizing multiple MS/MS product ions per lipid species not only improved the confidence of lipid identification but also enabled the determination of relative abundances of positional isomers in samples. Lipid class-based calibration curves were applied to interpolate lipid concentrations and guide sample dilution. Analytical validation was performed following FDA Bioanalytical Method Validation Guidance for Industry. We report repeatable and robust quantitation of 900 lipid species measured in NIST-SRM-1950 plasma, with over 700 lipids achieving inter-assay variability below 25%. To demonstrate proof of concept for biomarker discovery, we analyzed plasma from mice treated with a glucosylceramide synthase inhibitor, benzoxazole 1. We observed expected reductions in glucosylceramide levels in treated animals but, more notably, identified novel lipid biomarker candidates from the plasma lipidome. These data highlight the utility of this qualified lipidomic platform for enabling biological discovery.     

Extensive characterization of human tear fluid collected using different techniques unravels the presence of novel lipid amphiphiles

The tear film covers the anterior eye and the precise balance of its various constituting components is critical for maintaining ocular health. The composition of the tear film amphiphilic lipid sublayer, in particular, has largely remained a matter of contention due to the limiting concentrations of these lipid amphiphiles in tears that render their detection and accurate quantitation tedious. Using systematic and sensitive lipidomic approaches, we validated different tear collection techniques and report the most comprehensive human tear lipidome to date; comprising more than 600 lipid species from 17 major lipid classes. Our study confers novel insights to the compositional details of the existent tear film model, in particular the disputable amphiphilic lipid sublayer constituents, by demonstrating the presence of cholesteryl sulfate, O-acyl-ω-hydroxyfatty acids, and various sphingolipids and phospholipids in tears. The discovery and quantitation of the relative abundance of various tear lipid amphiphiles reported herein are expected to have a profound impact on the current understanding of the existent human tear film model.     

Top-Down Lipidomics Reveals Ether Lipid Deficiency in Blood Plasma of Hypertensive Patients

Background

Dyslipoproteinemia, obesity and insulin resistance are integrative constituents of the metabolic syndrome and are major risk factors for hypertension. The objective of this study was to determine whether hypertension specifically affects the plasma lipidome independently and differently from the effects induced by obesity and insulin resistance.

Methodology/Principal Findings

We screened the plasma lipidome of 19 men with hypertension and 51 normotensive male controls by top-down shotgun profiling on a LTQ Orbitrap hybrid mass spectrometer. The analysis encompassed 95 lipid species of 10 major lipid classes. Obesity resulted in generally higher lipid load in blood plasma, while the content of tri- and diacylglycerols increased dramatically. Insulin resistance, defined by HOMA-IR >3.5 and controlled for BMI, had little effect on the plasma lipidome. Importantly, we observed that in blood plasma of hypertensive individuals the overall content of ether lipids decreased. Ether phosphatidylcholines and ether phosphatidylethanolamines, that comprise arachidonic (20∶4) and docosapentaenoic (22∶5) fatty acid moieties, were specifically diminished. The content of free cholesterol also decreased, although conventional clinical lipid homeostasis indices remained unaffected.

Conclusions/Significance

Top-down shotgun lipidomics demonstrated that hypertension is accompanied by specific reduction of the content of ether lipids and free cholesterol that occurred independently of lipidomic alterations induced by obesity and insulin resistance. These results may form the basis for novel preventive and dietary strategies alleviating the severity of hypertension.     

A Lipidomic Study of the Effects of N-methyl-N＇-nitro-N-nitrosoguanidine on Sphingomyelin Metabolism

Systems biology is a new and rapidly developing research area in which, by quantitatively describing the interaction among all the individual components of a cell, a systems-level understanding of a biological response can be achieved. Therefore, it requires high-throughput measurement technologies for biological molecules, such as genomic and proteomic approaches for DNA/RNA and protein, respectively.Recently, a new concept, lipidomics, which utilizes the mass spectrometry （MS） method for lipid analysis,has been proposed. Using this lipidomic approach, the effects of N-methyl-N＇-nitro-N-nitrosoguanidine （MNNG） on sphingomyelin metabolism, a major class of sphingolipids, were evaluated. Sphingomyelin molecules were extracted from cells and analyzed by matrix-assisted laser desorption ionization-time of flight MS. It was found that MNNG induced profound changes in sphingomyelin metabolism, including the appearance of some new sphingomyelin species and the disappearance of some others, and the concentrations of several sphingomyelin species also changed. This was accompanied by the redistribution of acid sphingomyelinase （ASM）, a key player in sphingomyelin metabolism. On the other hand, imipramine, an inhibitor of ASM,caused the accumulation of sphingomyelin. It also prevented some of the effects of MNNG, as well as the redistribution of ASM. Taken together, these data suggested that the lipidomic approach is highly effective for the systematic analysis of cellular lipids metabolism.     

A Lipidomic Study of the Effects of N-methyl-N′-nitro-N-nitrosoguanidine on Sphingomyelin Metabolism

Systems biology is a new and rapidly developing research area in which,by quantitativelydescribing the interaction among all the individual components of a cell,a systems-level understanding of abiological response can be achieved.Therefore,it requires high-throughput measurement technologies forbiological molecules,such as genomic and proteomic approaches for DNA/RNA and protein,respectively.Recently,a new concept,lipidomics,which utilizes the mass spectrometry(MS)method for lipid analysis,has been proposed.Using this lipidomic approach,the effects of N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)on sphingomyelin metabolism,a major class of sphingolipids,were evaluated.Sphingomyelin moleculeswere extracted from cells and analyzed by matrix-assisted laser desorption ionization-time of flight MS.Itwas found that MNNG induced profound changes in sphingomyelin metabolism,including the appearance ofsome new sphingomyelin species and the disappearance of some others,and the concentrations of severalsphmgomyelin species also changed.This was accompanied by the redistribution of acid sphingomyelinase(ASM),a key player in sphingomyelin metabolism.On the other hand,imipramine,an inhibitor of ASM,caused the accumulation of sphingomyelin.It also prevented some of the effects of MNNG,as well as theredistribution of ASM.Taken together,these data suggested that the lipidomic approach is highly effectivefor the systematic analysis of cellular lipids metabolism.     

Lipidomic analysis of lipid droplets from murine hepatocytes reveals distinct signatures for nutritional stress

Liver steatosis can be induced by fasting or high-fat diet. We investigated by lipidomic analysis whether such metabolic states are reflected in the lipidome of hepatocyte lipid droplets (LDs) from mice fed normal chow diet (FED), fasted (FAS), or fed a high-fat diet (HFD). LC-MS/MS at levels of lipid species profiles and of lipid molecular species uncovered a FAS phenotype of LD enriched in triacylglycerol (TG) molecular species with very long-chain (VLC)-PUFA residues and an HFD phenotype with less unsaturated TG species in addition to characteristic lipid marker species. Nutritional stress did not result in dramatic structural alterations in diacylglycerol (DG) and phospholipid (PL) classes. Moreover, molecular species of bulk TG and of DG indicated concomitant de novo TG synthesis and lipase-catalyzed degradation to be active in LDs. DG species with VLC-PUFA residues would be preferred precursors for phosphatidylcholine (PC) species, the others for TG molecular species. In addition, molecular species of PL classes fitted the hepatocyte Kennedy and phosphatidylethanolamine methyltransferase pathways. We demonstrate that lipidomic analysis of LDs enables phenotyping of nutritional stress. TG species are best suited for such phenotyping, whereas structural analysis of TG, DG, and PL molecular species provides metabolic insights.     

Accumulation of raft lipids in T‐cell plasma membrane domains engaged in TCR signalling

Activating stimuli for T lymphocytes are transmitted through plasma membrane domains that form at T‐cell antigen receptor (TCR) signalling foci. Here, we determined the molecular lipid composition of immunoisolated TCR activation domains. We observed that they accumulate cholesterol, sphingomyelin and saturated phosphatidylcholine species as compared with control plasma membrane fragments. This provides, for the first time, direct evidence that TCR activation domains comprise a distinct molecular lipid composition reminiscent of liquid‐ordered raft phases in model membranes. Interestingly, TCR activation domains were also enriched in plasmenyl phosphatidylethanolamine and phosphatidylserine. Modulating the T‐cell lipidome with polyunsaturated fatty acids impaired the plasma membrane condensation at TCR signalling foci and resulted in a perturbed molecular lipid composition. These results correlate the accumulation of specific molecular lipid species with the specific plasma membrane condensation at sites of TCR activation and with early TCR activation responses.     

RNAi Transfection Results in Lipidome Changes

RNAi experiments are ubiquitously used in cell biology and are achieved by transfection of small interfering RNAs (siRNAs) into cells using a transfection reagent. These results in knock‐down of proteins of interest, and the phenotypic consequences are then analyzed. It is reported here that two common RNA interference (RNAi) transfection reagents, DharmaFECT 1 and INTERFERin, in mock transfections using non‐targeting siRNAs, cause alterations in the lipidome of HeLa cells. Some lipids change in response to both, presumably chemically different, transfection reagents, while other lipid species change only in response to one of the reagents. While the functional implications of these lipidomic alterations remain to be investigated, the authors' experiments suggest that it is important to use appropriate mock transfection controls during RNAi experiments, ideally complemented by an orthogonal perturbation, especially when investigating membrane‐associated phenomena.     

Quantification of sulfatides and lysosulfatides in tissues and body fluids by liquid chromatography-tandem mass spectrometry

Sulfatides are found in brain as components of myelin, oligodendrocytes, and neurons but are also present in various visceral tissues. Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disorder caused by a deficiency of arylsulfatase A, leading to severe white matter disease due to the accumulation of sulfatides and lysosulfatides. To study the physiological role of sulfatides, accessible and sensitive quantitative methods are required. We developed a sensitive LC/MS/MS method to quantify total sulfatide and lysosulfatide content as well as individual molecular species in urine and plasma from MLD patients and plasma and tissues from an MLD mouse model. Our results demonstrate that the method can quantify a wide range of sulfatide concentrations and can be used to quantify total sulfatide content and levels of individual molecular species of sulfatides in tissues, cells, and body fluids. Even though plasma sulfatides and lysosulfatides would seem attractive candidate biomarkers that could possibly correlate with the severity of MLD and be of use to monitor the effects of therapeutic intervention, our results indicate that it is unlikely that the determination of these storage products in plasma will be useful in this respect.     

Simultaneous extraction and rapid visualization of peptidomic and lipidomic body fluids fingerprints using mesoporous aluminosilicate and MALDI‐TOF MS

Herein we report the use of mesoporous aluminosilicate (MPAS) for the simultaneous extraction of peptides and lipids from complex body fluids such as human plasma and synovial fluid. We show that MPAS particles, given their mesostructural features with nanometric pore size and high surface area, are an efficient device for simultaneous extraction of peptidome and lipidome from as little as a few microliters of body fluids. The peptides and the lipids, selected and enriched by MPAS particles and rapidly visualized by MALDI‐TOF MS, could form part of a diagnostic profile of the “peptidome” and the “lipidome” of healthy versus diseased subjects in comparative studies. The ability of this approach to rapidly reveal the overall pattern of changes in both lipidome and peptidome signatures of complex biofluids could be of valuable interest for handling large numbers of samples required in ‐omics studies for the purpose of finding novel biomarkers.     

A Protective Lipidomic Biosignature Associated with a Balanced Omega-6/Omega-3 Ratio in fat-1 Transgenic Mice

A balanced omega-6/omega-3 polyunsaturated fatty acid (PUFA) ratio has been linked to health benefits and the prevention of many chronic diseases. Current dietary intervention studies with different sources of omega-3 fatty acids (omega-3) lack appropriate control diets and carry many other confounding factors derived from genetic and environmental variability. In our study, we used the fat-1 transgenic mouse model as a proxy for long-term omega-3 supplementation to determine, in a well-controlled manner, the molecular phenotype associated with a balanced omega-6/omega-3 ratio. The fat-1 mouse can convert omega-6 to omega-3 PUFAs, which protect against a wide variety of diseases including chronic inflammatory diseases and cancer. Both wild-type (WT) and fat-1 mice were subjected to an identical diet containing 10% corn oil, which has a high omega-6 content similar to that of the Western diet, for a six-month duration. We used a multi-platform lipidomic approach to compare the plasma lipidome between fat-1 and WT mice. In fat-1 mice, an unbiased profiling showed a significant increase in the levels of unesterified eicosapentaenoic acid (EPA), EPA-containing cholesteryl ester, and omega-3 lysophosphospholipids. The increase in omega-3 lipids is accompanied by a significant reduction in omega-6 unesterified docosapentaenoic acid (omega-6 DPA) and DPA-containing cholesteryl ester as well as omega-6 phospholipids and triacylglycerides. Targeted lipidomics profiling highlighted a remarkable increase in EPA-derived diols and epoxides formed via the cytochrome P450 (CYP450) pathway in the plasma of fat-1 mice compared with WT mice. Integration of the results of untargeted and targeted analyses has identified a lipidomic biosignature that may underlie the healthful phenotype associated with a balanced omega-6/omega-3 ratio, and can potentially be used as a circulating biomarker for monitoring the health status and the efficacy of omega-3 intervention in humans.     

Lipidomic analysis of epithelial corneal cells following hyperosmolarity and benzalkonium chloride exposure: New insights in dry eye disease

Dry eye disease (DED) is a multifactorial chronic inflammatory disease of the ocular surface characterized by tear film instability, hyperosmolarity, cell damage and inflammation. Hyperosmolarity is strongly established as the core mechanism of the DED. Benzalkonium chloride (BAK) - a quaternary ammonium salt commonly used in eye drops for its microbicidal properties - is well known to favor the onset of DED. Currently, little data are available regarding lipid metabolism alteration in ocular surface epithelial cells in the course of DED. Our aim was to explore the effects of benzalkonium chloride or hyperosmolarity exposure on the human corneal epithelial (HCE) cell lipidome, two different conditions used as in vitro models of DED. For this purpose, we performed a lipidomic analysis using UPLC-HRMS-ESI+/−. Our results demonstrated that BAK or hyperosmolarity induced important modifications in HCE lipidome including major changes in sphingolipids, glycerolipids and glycerophospholipids. For both exposures, an increase in ceramide was especially exhibited. Hyperosmolarity specifically induced triglyceride accumulation resulting in lipid droplet formation. Conversely, BAK induced an increase in lysophospholipids and a decrease in phospholipids. This lipidomic study highlights the lipid changes involved in inflammatory responses following BAK or hyperosmolarity exposures. Thereby, lipid research appears of great interest, as it could lead to the discovery of new biomarkers and therapeutic targets for the diagnosis and treatment of dry eye disease.     

Detection and confirmation of serum lipid biomarkers for preeclampsia using direct infusion mass spectrometry

Despite substantial research, the early diagnosis of preeclampsia remains elusive. Lipids are now recognized to be involved in regulation and pathophysiology of some disease. Shotgun lipidomic studies were undertaken to determine whether serum lipid biomarkers exist that predict preeclampsia later in the same in pregnancy. A discovery study was performed using sera collected at 12–14 weeks pregnancy from 27 controls with uncomplicated pregnancies and 29 cases that later developed preeclampsia. Lipids were extracted and analyzed by direct infusion into a TOF mass spectrometer. MS signals, demonstrating apparent differences were selected, their abundances determined, and statistical differences tested. Statistically significant lipid markers were reevaluated in a second confirmatory study having 43 controls and 37 preeclampsia cases. Multi-marker combinations were developed using those lipid biomarkers confirmed in the second study. The initial study detected 45 potential preeclampsia markers. Of these, 23 markers continued to be statistically significant in the second confirmatory set. Most of these markers, representing several lipid classes, were chemically characterized, typically providing lipid class and potential molecular components using MS². Several multi-marker panels with areas under the curve >0.85 and high predictive values were developed. Developed panels of serum lipidomic biomarkers appear to be able to identify most women at risk for preeclampsia in a given pregnancy at 12–14 weeks gestation.     

Alterations in lipid homeostasis of mouse dorsal root ganglia induced by apolipoprotein E deficiency: a shotgun lipidomics study

One of the fundamental goals of lipidomics research is to identify the linkage of an individual gene with a given lipidome, thereby revealing the role of that gene in lipid metabolism, transport, and homeostasis. In this study, we have identified four apolipoprotein E (apoE)-induced alterations in the lipidome of mouse dorsal root ganglia (DRG) through utilizing the technology of shotgun lipidomics. First, apoE mediates sulfatide mass content in mouse DRG, which is comparable to its role in the CNS. Second, apoE contributes to galactosylceramide and ceramide homeostasis in mouse DRG. Third, apoE significantly modulates cholesterol levels in mouse DRG. The latter two functions of apoE are distinct from those in the CNS. Finally, mice null for apoE have dramatically less triacylglycerol mass content in DRG which are opposite to the effects observed in the peripheral organs and vascular system. Collectively, this study identifies the specific alterations in the DRG lipidome induced by apoE knockout and suggests the potential roles of apoE in lipid transport and homeostasis in a tissue specific manner, thereby providing insights into the biochemical mechanisms underlying the functions of apoE in the PNS.     

Comprehensive analysis of the major lipid classes in sebum by rapid resolution high-performance liquid chromatography and electrospray mass spectrometry

Sebum is a complex lipid mixture that is synthesized in sebaceous glands and excreted on the skin surface. The purpose of this study was the comprehensive detection of the intact lipids that compose sebum. These lipids exist as a broad range of chemical structures and concentrations. Sebum was collected with SebuTape^TM from the foreheads of healthy donors, and then separated by HPLC on a C8 stationary phase with sub 2 µm particle size. This HPLC method provided high resolution and excellent reproducibility of retention times (RT). Compound mining was performed with time of flight (TOF) and triple quadrupole (QqQ) mass spectrometers (MS), which allowed for the classification of lipids according to their elemental composition, degree of unsaturation, and MS/MS fragmentation. The combination of the two MS systems detected 95 and 29 families of triacylglycerols (TAG) and diacylglycerols (DAG), respectively. Assignment was carried out regardless of positional isomerism. Among the wax esters (WE), 28 species were found to contain the 16:1 fatty acyl moiety. This method was suitable for the simultaneous detection of squalene and its oxygenated derivative. A total of 9 cholesterol esters (CE) were identified and more than 48 free fatty acids (FFA) were detected in normal sebum. The relative abundance of each individual lipid within its own chemical class was determined for 12 healthy donors. In summary, this method provided the first characterization of the features and distribution of intact components of the sebum lipidome.