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1.  Human Plasma Lipidome Is Pleiotropically Associated with Cardiovascular Risk Factors and Death 
Background
Cardiovascular disease (CVD) is the most common cause of death in the United States and is associated with a high economic burden. Prevention of CVD focuses on controlling or improving the lipid profile of patients at risk. The human lipidome is made up of thousands of ubiquitous lipid species. By studying biologically simple canonical lipid species, we investigated whether the lipidome is genetically redundant and whether its genetic influences can provide clinically relevant clues of CVD risk.
Methods and Results
We performed a genetic study of the human lipidome in 1,212 individuals from 42 extended Mexican American families. High-throughput mass spectrometry enabled rapid capture of precise lipidomic profiles, providing 319 unique species. Using variance-component based heritability analyses and bivariate trait analyses, we detected significant genetic influences on each lipid assayed. Median heritability of the plasma lipid species was 0.37. Hierarchical clustering based on complex genetic correlation patterns identified 12 genetic clusters that characterized the plasma lipidome. These genetic clusters were differentially but consistently associated with risk factors of CVD, including central obesity, obesity, type 2 diabetes, raised serum triglycerides and metabolic syndrome. Also these clusters consistently predicted occurrence of cardiovascular deaths during follow-up.
Conclusions
The human plasma lipidome is heritable. Shared genetic influences reduce the dimensionality of the human lipidome into clusters that are associated with risk factors of CVD.
doi:10.1161/CIRCGENETICS.114.000600
PMCID: PMC4270876  PMID: 25363705
lipids; genetics; cardiovascular disease; family study; genetic correlation
2.  Gender, Contraceptives and Individual Metabolic Predisposition Shape a Healthy Plasma Lipidome 
Scientific Reports  2016;6:27710.
Lipidomics of human blood plasma is an emerging biomarker discovery approach that compares lipid profiles under pathological and physiologically normal conditions, but how a healthy lipidome varies within the population is poorly understood. By quantifying 281 molecular species from 27 major lipid classes in the plasma of 71 healthy young Caucasians whose 35 clinical blood test and anthropometric indices matched the medical norm, we provided a comprehensive, expandable and clinically relevant resource of reference molar concentrations of individual lipids. We established that gender is a major lipidomic factor, whose impact is strongly enhanced by hormonal contraceptives and mediated by sex hormone-binding globulin. In lipidomics epidemiological studies should avoid mixed-gender cohorts and females taking hormonal contraceptives should be considered as a separate sub-cohort. Within a gender-restricted cohort lipidomics revealed a compositional signature that indicates the predisposition towards an early development of metabolic syndrome in ca. 25% of healthy male individuals suggesting a healthy plasma lipidome as resource for early biomarker discovery.
doi:10.1038/srep27710
PMCID: PMC4906355  PMID: 27295977
3.  The LUX Score: A Metric for Lipidome Homology 
PLoS Computational Biology  2015;11(9):e1004511.
A lipidome is the set of lipids in a given organism, cell or cell compartment and this set reflects the organism’s synthetic pathways and interactions with its environment. Recently, lipidomes of biological model organisms and cell lines were published and the number of functional studies of lipids is increasing. In this study we propose a homology metric that can quantify systematic differences in the composition of a lipidome. Algorithms were developed to 1. consistently convert lipids structure into SMILES, 2. determine structural similarity between molecular species and 3. describe a lipidome in a chemical space model. We tested lipid structure conversion and structure similarity metrics, in detail, using sets of isomeric ceramide molecules and chemically related phosphatidylinositols. Template-based SMILES showed the best properties for representing lipid-specific structural diversity. We also show that sequence analysis algorithms are best suited to calculate distances between such template-based SMILES and we adjudged the Levenshtein distance as best choice for quantifying structural changes. When all lipid molecules of the LIPIDMAPS structure database were mapped in chemical space, they automatically formed clusters corresponding to conventional chemical families. Accordingly, we mapped a pair of lipidomes into the same chemical space and determined the degree of overlap by calculating the Hausdorff distance. We named this metric the ‘Lipidome jUXtaposition (LUX) score’. First, we tested this approach for estimating the lipidome similarity on four yeast strains with known genetic alteration in fatty acid synthesis. We show that the LUX score reflects the genetic relationship and growth temperature better than conventional methods although the score is based solely on lipid structures. Next, we applied this metric to high-throughput data of larval tissue lipidomes of Drosophila. This showed that the LUX score is sufficient to cluster tissues and determine the impact of nutritional changes in an unbiased manner, despite the limited information on the underlying structural diversity of each lipidome. This study is the first effort to define a lipidome homology metric based on structures that will enrich functional association of lipids in a similar manner to measures used in genetics. Finally, we discuss the significance of the LUX score to perform comparative lipidome studies across species borders.
Author Summary
Because of their role in health and disease, lipids are often the focus of biochemical studies. Advances in analytical biochemistry have made it possible to detect all the lipids from a cell, tissue or organism (termed lipidome). Much of this research is based on model organisms, but it is difficult to transfer results from a fruit fly or yeast to human biochemistry. A central problem is that there is no agreed-upon method for comparing lipidomes. We have developed the LUX score, which enables us to determine the homology between lipidomes. All constituent lipids are first embedded in a chemical space according to their similarity to each other. When we treat all lipids as points in such a space, one can overlay different lipidomes and estimate their differences. We expect that this kind of metric will be useful for translating findings from model organisms to human diseases and in understanding fundamental biological processes.
doi:10.1371/journal.pcbi.1004511
PMCID: PMC4578897  PMID: 26393792
4.  A novel strategy for the comprehensive analysis of the biomolecular composition of isolated plasma membranes 
A methodology for rapid, high-purity isolation of plasma membranes using superparamagnetic nanoparticles is described. The method is illustrated with high-resolution proteomic, glycomic and lipidomic analyses of presenilin-deficient cells.
We present a novel strategy based on cationic phospholipids-coated superparamagnetic nanoparticles (SPMNPs) to isolate plasma membranes with very high purity and at a preparative scale.The SPMNP-based isolation method is compatible with subsequent analysis of the biomolecular composition of plasma membranes, including proteomics, lipidomics and N-glycomics analysis.A comparative ‘omics' analysis of plasma membranes from wild-type, presenilin-deficient and human presenilin-1-rescued fibroblasts revealed convergent changes in proteins and lipids, suggesting an underlying endosomal transport defect.Our methodology allows for the systematic set-up of comprehensive plasma membrane inventories: alterations in the composition of the cell surface may potentially identify novel biomarkers or drug targets.
One of the major goals of this paper was to establish a robust method for plasma membrane (PM) isolation in order to perform a full analysis of their biomolecular composition. Using thermal decomposition, we manufactured superparamagnetic nanoparticles (SPMNPs) that we rendered water soluble and monodisperse by subsequent coupling of NH2 phospholipids. When incubated with cell monolayers, these cationic phospholipids-SPMNPs remain predominately localized at the cell surface. We applied these unexpected feature of phospholipids-SPMNPs to establish a novel protocol to isolate high yields of highly pure PMs. Due to the superb quality and quantity of isolated PM fractions, we could perform a comprehensive and comparative biomolecular profiling on this subcellular compartment that included proteomics, N-glycoproteomics, lipidomics and N-glycan profiling. This method was subsequently applied to compare the biomolecular composition of PMs isolated from wild-type and presenilin-deficient and human presenilin-1-rescued mouse embryonic fibroblasts (MEFs). For the first time, we succeeded in identifying convergent changes in the biomolecular composition of the cell surface caused by presenilin gene deficiency. Furthermore, the observed proteomic/cholesterol changes were restored in presenilin-deficient MEFs rescued with the human presenilin-1 ortholog. These (subtle) changes in protein and lipid composition suggest an underlying endosomal transport defect in presenilin-deficient cell lines that is the subject of ongoing research.
Moreover, and extending the versatility of our method, we could show that our PM isolations are compatible with fluorophore-assisted carbohydrate electrophoresis (FACE), allowing for N-glycan profiling of the cell surface. Hitherto, the N-glycan composition was measured on total cell extracts where the sensitivity to detect mature N-glycan chains is significantly hampered by the higher abundances of intracellular immature N-glycan intermediates. Finally, using the γ-secretase protein complex as a model, we confirm that we can study the activity and composition of active protein complexes in their native membrane environment. Our strategy incorporates for the first time most available omics analyses and this on a single isolated membrane compartment. As such, it allows for the monitoring and identification of systematic changes at the cell surface, for instance during differentiation and polarization or as a consequence of environmental insults, ER-stress, apoptotic stimuli and altered lipogenesis. The identification of alterations in the PM protein and lipid composition, as they occur as a consequence of disease, is therefore of paramount importance in several fields of experimental medicine, including immunology, cancer and stem cell research. Our methodology provides also a foundation to systematically start collecting PM ‘fingerprints' of an increasing number of cells. The resulting integrated and comprehensive databases may become the starting point of a ‘subcellular systems biology' approach of the cell's limiting membrane. Since PM proteins provide many pathological relevant biomarkers representing two-thirds of the currently used drug targets, this novel technology has great potential for biomedical and pharmaceutical applications.
We manufactured a novel type of lipid-coated superparamagnetic nanoparticles that allow for a rapid isolation of plasma membranes (PMs), enabling high-resolution proteomic, glycomic and lipidomic analyses of the cell surface. We used this technology to characterize the effects of presenilin knockout on the PM composition of mouse embryonic fibroblasts. We found that many proteins are selectively downregulated at the cell surface of presenilin knockout cells concomitant with lowered surface levels of cholesterol and certain sphingomyelin species, indicating defects in specific endosomal transport routes to and/or from the cell surface. Snapshots of N-glycoproteomics and cell surface glycan profiling further underscored the power and versatility of this novel methodology. Since PM proteins provide many pathologically relevant biomarkers representing two-thirds of the currently used drug targets, this novel technology has great potential for biomedical and pharmaceutical applications.
doi:10.1038/msb.2011.74
PMCID: PMC3261717  PMID: 22027552
eukaryotic cell systems; glycomics; lipidomics; presenilin, proteomics
5.  A Protective Lipidomic Biosignature Associated with a Balanced Omega-6/Omega-3 Ratio in fat-1 Transgenic Mice 
PLoS ONE  2014;9(4):e96221.
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.
doi:10.1371/journal.pone.0096221
PMCID: PMC3997567  PMID: 24760204
6.  Plasma Lysophosphatidylcholine Levels Are Reduced in Obesity and Type 2 Diabetes 
PLoS ONE  2012;7(7):e41456.
Background
Obesity and type 2 diabetes (T2DM) are associated with increased circulating free fatty acids and triacylglycerols. However, very little is known about specific molecular lipid species associated with these diseases. In order to gain further insight into this, we performed plasma lipidomic analysis in a rodent model of obesity and insulin resistance as well as in lean, obese and obese individuals with T2DM.
Methodology/Principal Findings
Lipidomic analysis using liquid chromatography coupled to mass spectrometry revealed marked changes in the plasma of 12 week high fat fed mice. Although a number of triacylglycerol and diacylglycerol species were elevated along with of a number of sphingolipids, a particularly interesting finding was the high fat diet (HFD)-induced reduction in lysophosphatidylcholine (LPC) levels. As liver, skeletal muscle and adipose tissue play an important role in metabolism, we next determined whether the HFD altered LPCs in these tissues. In contrast to our findings in plasma, only very modest changes in tissue LPCs were noted. To determine when the change in plasma LPCs occurred in response to the HFD, mice were studied after 1, 3 and 6 weeks of HFD. The HFD caused rapid alterations in plasma LPCs with most changes occurring within the first week. Consistent with our rodent model, data from our small human cohort showed a reduction in a number of LPC species in obese and obese individuals with T2DM. Interestingly, no differences were found between the obese otherwise healthy individuals and the obese T2DM patients.
Conclusion
Irrespective of species, our lipidomic profiling revealed a generalized decrease in circulating LPC species in states of obesity. Moreover, our data indicate that diet and adiposity, rather than insulin resistance or diabetes per se, play an important role in altering the plasma LPC profile.
doi:10.1371/journal.pone.0041456
PMCID: PMC3405068  PMID: 22848500
7.  Metabolic Regulation in Progression to Autoimmune Diabetes 
PLoS Computational Biology  2011;7(10):e1002257.
Recent evidence from serum metabolomics indicates that specific metabolic disturbances precede β-cell autoimmunity in humans and can be used to identify those children who subsequently progress to type 1 diabetes. The mechanisms behind these disturbances are unknown. Here we show the specificity of the pre-autoimmune metabolic changes, as indicated by their conservation in a murine model of type 1 diabetes. We performed a study in non-obese prediabetic (NOD) mice which recapitulated the design of the human study and derived the metabolic states from longitudinal lipidomics data. We show that female NOD mice who later progress to autoimmune diabetes exhibit the same lipidomic pattern as prediabetic children. These metabolic changes are accompanied by enhanced glucose-stimulated insulin secretion, normoglycemia, upregulation of insulinotropic amino acids in islets, elevated plasma leptin and adiponectin, and diminished gut microbial diversity of the Clostridium leptum group. Together, the findings indicate that autoimmune diabetes is preceded by a state of increased metabolic demands on the islets resulting in elevated insulin secretion and suggest alternative metabolic related pathways as therapeutic targets to prevent diabetes.
Author Summary
We have recently found that distinct metabolic disturbances precede β-cell autoimmunity in children who later progress to type 1 diabetes (T1D). Here we performed a murine study using non-obese diabetic (NOD) mice that recapitulated the protocol used in human, followed up by independent studies where NOD mice were studied in relation to risk of diabetes progression. We found that young female NOD mice who later progress to autoimmune diabetes exhibit the same lipidomic pattern as prediabetic children. These metabolic changes are accompanied by enhanced glucose-stimulated insulin secretion, upregulation of insulinotropic amino acids in islets, elevated plasma leptin and adiponectin, and diminished gut microbial diversity of the Clostridium leptum subgroup. The metabolic phenotypes observed in our study could be relevant as end points for studies investigating T1D pathogenesis and/or responses to interventions. By proceeding from a clinical study via metabolomics and modeling to an experimental model using a similar study design, then evolving further to tissue-specific studies, we hereby also present a conceptually novel approach to reversed translation that may be useful in future therapeutic studies in the context of prevention and treatment of T1D as well as of other diseases characterized by long prodromal periods.
doi:10.1371/journal.pcbi.1002257
PMCID: PMC3203065  PMID: 22046124
8.  Shotgun Lipidomics by Sequential Precursor Ion Fragmentation on a Hybrid Quadrupole Time-of-Flight Mass Spectrometer 
Metabolites  2012;2(1):195-213.
Shotgun lipidomics has evolved into a myriad of multi-dimensional strategies for molecular lipid characterization, including bioinformatics tools for mass spectrum interpretation and quantitative measurements to study systems-lipidomics in complex biological extracts. Taking advantage of spectral mass accuracy, scan speed and sensitivity of improved quadrupole linked time-of-flight mass analyzers, we developed a bias-free global lipid profiling acquisition technique of sequential precursor ion fragmentation called MS/MSALL. This generic information-independent tandem mass spectrometry (MS) technique consists of a Q1 stepped mass isolation window through a set mass range in small increments, fragmenting and recording all product ions and neutral losses. Through the accurate MS and MS/MS information, the molecular lipid species are resolved, including distinction of isobaric and isomeric species, and composed into more precise lipidomic outputs. The method demonstrates good reproducibility and at least 3 orders of dynamic quantification range for isomeric ceramides in human plasma. More than 400 molecular lipids in human plasma were uncovered and quantified in less than 12 min, including acquisitions in both positive and negative polarity modes. We anticipate that the performance of sequential precursor ion fragmentation both in quality and throughput will lead to the uncovering of new avenues throughout the biomedical research community, enhance biomarker discovery and provide novel information target discovery programs as it will prospectively shed new insight into affected metabolic and signaling pathways.
doi:10.3390/metabo2010195
PMCID: PMC3901199  PMID: 24957374
MS/MSALL; lipidomics; lipid; QTOF; mass spectrometry
9.  Mapping the Human Platelet Lipidome Reveals Cytosolic Phospholipase A2 as a Regulator of Mitochondrial Bioenergetics during Activation 
Cell Metabolism  2016;23(5):930-944.
Summary
Human platelets acutely increase mitochondrial energy generation following stimulation. Herein, a lipidomic circuit was uncovered whereby the substrates for this are exclusively provided by cPLA2, including multiple fatty acids and oxidized species that support energy generation via β-oxidation. This indicates that acute lipid membrane remodeling is required to support energetic demands during platelet activation. Phospholipase activity is linked to energy metabolism, revealing cPLA2 as a central regulator of both lipidomics and energy flux. Using a lipidomic approach (LipidArrays), we also estimated the total number of lipids in resting, thrombin-activated, and aspirinized platelets. Significant diversity between genetically unrelated individuals and a wealth of species was revealed. Resting platelets demonstrated ∼5,600 unique species, with only ∼50% being putatively identified. Thrombin elevated ∼900 lipids >2-fold with 86% newly appearing and 45% inhibited by aspirin supplementation, indicating COX-1 is required for major activation-dependent lipidomic fluxes. Many lipids were structurally identified. With ∼50% of the lipids being absent from databases, a major opportunity for mining lipids relevant to human health and disease is presented.
Graphical Abstract
Highlights
•The complete lipidome of resting and agonist-activated human platelets is presented•Low-dose aspirin supplementation profoundly affects the platelet lipidome•Significant diversity in the lipidomes of three unrelated individuals is seen•Cytosolic phospholipase A2 provides lipids for mitochondrial oxygen consumption
Slatter et al. characterize the lipidomic network of human platelets. Besides the characterization of nearly 200 oxidized species, the resource shows that remodeling of the membrane via phospholipase activity provides energy substrates for respiration. The findings demonstrate a direct link between innate immunity and mitochondrial bioenergetics in human platelets.
doi:10.1016/j.cmet.2016.04.001
PMCID: PMC4873619  PMID: 27133131
10.  Characterization and direct quantitation of sphingoid base-1-phosphates from lipid extracts: a shotgun lipidomics approach 
Journal of lipid research  2006;47(8):1865-1873.
Here, we have extended shotgun lipidomics for the characterization and quantitation of sphingosine-1-phosphate (S1P) and dihydrosphingosine-1-phosphate (DHS1P) in crude lipid extracts in the presence of ammonium hydroxide by using precursor ion scanning of m/z 79.0 (corresponding to [PO3]−) in the negative-ion mode. It is demonstrated that a broad linear dynamic range for the quantitation of both S1P and DHS1P and a detection limit at low amol/μl concentration are achieved using this approach. The developed method for the quantitation of sphingoid base-1-phosphates is generally simpler and more efficient than other previously published methods. Multiple factors influencing the quantitation of sphingoid base-1-phosphates, including ion suppression, extraction efficiency, and potential overlapping with other molecular species, were examined extensively and/or are discussed. Mass levels of S1P and DHS1P in multiple biological samples, including human plasma, mouse plasma, and mouse brain tissues (e.g., cortex, cerebellum, spinal cord, and brain stem), were determined by the developed methodology. Accordingly, this technique, as a new addition to shotgun lipidomics technology, will be extremely useful for understanding the pathways of sphingolipid metabolism and for exploring the important roles of sphingoid base-1-phosphates in a wide range of physiological and pathological studies.—Jiang, X., and X. Han. Characterization and direct quantitation of sphingoid base-1-phosphates from lipid extracts: a shotgun lipidomics approach.
doi:10.1194/jlr.D600012-JLR200
PMCID: PMC2141545  PMID: 16682747
dihydrosphingosine-1-phosphate; electrospray ionization mass spectrometry; brain tissues; sphingolipid metabolism
11.  A reversed-phase capillary ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) method for comprehensive top-down/bottom-up lipid profiling 
Analytical and bioanalytical chemistry  2012;402(9):2923-2933.
Lipidomics is a critical part of metabolomics and aims to study all the lipids within a living system. We present here the development and evaluation of a sensitive capillary UPLC-MS method for comprehensive top-down/bottom-up lipid profiling. Three different stationary phases were evaluated in terms of peak capacity, linearity, reproducibility, and limit of quantification (LOQ) using a mixture of lipid standards representative of the lipidome. The relative standard deviations of the retention times and peak abundances of the lipid standards were 0.29% and 7.7%, respectively, when using the optimized method. The linearity was acceptable at >0.99 over 3 orders of magnitude, and the LOQs were sub-fmol. To demonstrate the performance of the method in the analysis of complex samples, we analyzed lipids extracted from a human cell line, rat plasma, and a model human skin tissue, identifying 446, 444, and 370 unique lipids, respectively. Overall, the method provided either higher coverage of the lipidome, greater measurement sensitivity, or both, when compared to other approaches of global, untargeted lipid profiling based on chromatography coupled with MS.
doi:10.1007/s00216-012-5773-5
PMCID: PMC3531553  PMID: 22354571
ultra-performance liquid chromatography (UPLC); tandem mass spectrometry (MS/MS); electrospray ionization (ESI); top-down/bottom-up lipid profiling
12.  Comparative Plasma Lipidome between Human and Cynomolgus Monkey: Are Plasma Polar Lipids Good Biomarkers for Diabetic Monkeys? 
PLoS ONE  2011;6(5):e19731.
Background
Non-human primates (NHP) are now being considered as models for investigating human metabolic diseases including diabetes. Analyses of cholesterol and triglycerides in plasma derived from NHPs can easily be achieved using methods employed in humans. Information pertaining to other lipid species in monkey plasma, however, is lacking and requires comprehensive experimental analysis.
Methodologies/Principal Findings
We examined the plasma lipidome from 16 cynomolgus monkey, Macaca fascicularis, using liquid chromatography coupled with mass spectrometry (LC/MS). We established novel analytical approaches, which are based on a simple gradient elution, to quantify polar lipids in plasma including (i) glycerophospholipids (phosphatidylcholine, PC; phosphatidylethanolamine, PE; phosphatidylinositol, PI; phosphatidylglycerol, PG; phosphatidylserine, PS; phosphatidic acid, PA); (ii) sphingolipids (sphingomyelin, SM; ceramide, Cer; Glucocyl-ceramide, GluCer; ganglioside mannoside 3, GM3). Lipidomic analysis had revealed that the plasma of human and cynomolgus monkey were of similar compositions, with PC, SM, PE, LPC and PI constituting the major polar lipid species present. Human plasma contained significantly higher levels of plasmalogen PE species (p<0.005) and plasmalogen PC species (p<0.0005), while cynomolgus monkey had higher levels of polyunsaturated fatty acyls (PUFA) in PC, PE, PS and PI. Notably, cynomolgus monkey had significantly lower levels of glycosphingolipids, including GluCer (p<0.0005) and GM3 (p<0.0005), but higher level of Cer (p<0.0005) in plasma than human. We next investigated the biochemical alterations in blood lipids of 8 naturally occurring diabetic cynomolgus monkeys when compared with 8 healthy controls.
Conclusions
For the first time, we demonstrated that the plasma of human and cynomolgus monkey were of similar compositions, but contained different mol distribution of individual molecular species. Diabetic monkeys exhibited decreased levels of sphingolipids, which are microdomain-associated lipids and are thought to be associated with insulin sensitivity. Significant increases in PG species, which are precursors for cardiolipin biosynthesis in mitochondria, were found in fasted diabetic monkeys (n = 8).
doi:10.1371/journal.pone.0019731
PMCID: PMC3087804  PMID: 21573191
13.  Integrated lipidomics and transcriptomic analysis of peripheral blood reveals significantly enriched pathways in type 2 diabetes mellitus 
BMC Medical Genomics  2013;6(Suppl 1):S12.
Background
Insulin resistance is a key element in the pathogenesis of type 2 diabetes mellitus. Plasma free fatty acids were assumed to mediate the insulin resistance, while the relationship between lipid and glucose disposal remains to be demonstrated across liver, skeletal muscle and blood.
Methods
We profiled both lipidomics and gene expression of 144 total peripheral blood samples, 84 from patients with T2D and 60 from healthy controls. Then, factor and partial least squares models were used to perform a combined analysis of lipidomics and gene expression profiles to uncover the bioprocesses that are associated with lipidomic profiles in type 2 diabetes.
Results
According to factor analysis of the lipidomic profile, several species of lipids were found to be correlated with different phenotypes, including diabetes-related C23:2CE, C23:3CE, C23:4CE, ePE36:4, ePE36:5, ePE36:6; race-related (African-American) PI36:1; and sex-related PE34:1 and LPC18:2. The major variance of gene expression profile was not caused by known factors and no significant difference can be directly derived from differential gene expression profile. However, the combination of lipidomic and gene expression analyses allows us to reveal the correlation between the altered lipid profile with significantly enriched pathways, such as one carbon pool by folate, arachidonic acid metabolism, insulin signaling pathway, amino sugar and nucleotide sugar metabolism, propanoate metabolism, and starch and sucrose metabolism. The genes in these pathways showed a good capability to classify diabetes samples.
Conclusion
Combined analysis of gene expression and lipidomic profiling reveals type 2 diabetes-associated lipid species and enriched biological pathways in peripheral blood, while gene expression profile does not show direct correlation. Our findings provide a new clue to better understand the mechanism of disordered lipid metabolism in association with type 2 diabetes.
doi:10.1186/1755-8794-6-S1-S12
PMCID: PMC3552685  PMID: 23369247
14.  Altered Sphingolipid Metabolism in Patients with Metastatic Pancreatic Cancer 
Biomolecules  2013;3(3):435-448.
Although numerous genetic mutations and amplifications have been identified in pancreatic cancer, much of the molecular pathogenesis of the disease remains undefined. While proteomic and transcriptomic analyses have been utilized to probe and characterize pancreatic tumors, lipidomic analyses have not been applied to identify perturbations in pancreatic cancer patient samples. Thus, we utilized a mass spectrometry-based lipidomic approach, focused towards the sphingolipid class of lipids, to quantify changes in human pancreatic cancer tumor and plasma specimens. Subgroup analysis revealed that patients with positive lymph node metastasis have a markedly higher level of ceramide species (C16:0 and C24:1) in their tumor specimens compared to pancreatic cancer patients without nodal disease or to patients with pancreatitis. Also of interest, ceramide metabolites, including phosphorylated (sphingosine- and sphinganine-1-phosphate) and glycosylated (cerebroside) species were elevated in the plasma, but not the pancreas, of pancreatic cancer patients with nodal disease. Analysis of plasma level of cytokine and growth factors revealed that IL-6, IL-8, CCL11 (eotaxin), EGF and IP10 (interferon inducible protein 10, CXCL10) were elevated in patients with positive lymph nodes metastasis, but that only IP10 and EGF directly correlated with several sphingolipid changes. Taken together, these data indicate that sphingolipid metabolism is altered in human pancreatic cancer and associated with advanced disease. Assessing plasma and/or tissue sphingolipids could potentially risk stratify patients in the clinical setting.
doi:10.3390/biom3030435
PMCID: PMC4030952  PMID: 24970174
sphingolipids; pancreatic cancer; ceramide; lipidomics; CXCL10
15.  Dietary fat and not calcium supplementation or dairy product consumption is associated with changes in anthropometrics during a randomized, placebo-controlled energy-restriction trial 
Insufficient calcium intake has been proposed to cause unbalanced energy partitioning leading to obesity. However, weight loss interventions including dietary calcium or dairy product consumption have not reported changes in lipid metabolism measured by the plasma lipidome.
Methods
The objective of this study was to determine the relationships between dairy product or supplemental calcium intake with changes in the plasma lipidome and body composition during energy restriction. A secondary objective of this study was to explore the relationships among calculated macronutrient composition of the energy restricted diet to changes in the plasma lipidome, and body composition during energy restriction. Overweight adults (n = 61) were randomized into one of three intervention groups including a deficit of 500kcal/d: 1) placebo; 2) 900 mg/d calcium supplement; and 3) 3-4 servings of dairy products/d plus a placebo supplement. Plasma fatty acid methyl esters of cholesterol ester, diacylglycerol, free fatty acids, lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine and triacylglycerol were quantified by capillary gas chromatography.
Results
After adjustments for energy and protein (g/d) intake, there was no significant effect of treatment on changes in weight, waist circumference or body composition. Plasma lipidome did not differ among dietary treatment groups. Stepwise regression identified correlations between reported intake of monounsaturated fat (% of energy) and changes in % lean mass (r = -0.44, P < 0.01) and % body fat (r = 0.48, P < 0.001). Polyunsaturated fat intake was associated with the % change in waist circumference (r = 0.44, P < 0.01). Dietary saturated fat was not associated with any changes in anthropometrics or the plasma lipidome.
Conclusions
Dairy product consumption or calcium supplementation during energy restriction over the course of 12 weeks did not affect plasma lipids. Independent of calcium and dairy product consumption, short-term energy restriction altered body composition. Reported dietary fat composition of energy restricted diets was associated with the degree of change in body composition in these overweight and obese individuals.
doi:10.1186/1743-7075-8-67
PMCID: PMC3204227  PMID: 21970320
16.  Top-Down Lipidomics Reveals Ether Lipid Deficiency in Blood Plasma of Hypertensive Patients 
PLoS ONE  2009;4(7):e6261.
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.
doi:10.1371/journal.pone.0006261
PMCID: PMC2705678  PMID: 19603071
17.  Statin action favors normalization of the plasma lipidome in the atherogenic mixed dyslipidemia of MetS: potential relevance to statin-associated dysglycemia[S] 
Journal of Lipid Research  2015;56(12):2381-2392.
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.
doi:10.1194/jlr.P061143
PMCID: PMC4655992  PMID: 26486974
lipidomics; obesity; plasmalogens; cholesterol; omega-3 fatty acids; pitavastatin; metabolic syndrome
18.  Effect of probiotic Lactobacillus rhamnosus GG intervention on global serum lipidomic profiles in healthy adults 
AIM: To investigate the effect of three weeks’ intervention with a probiotic Lactobacillus rhamnosus GG (LGG) bacteria on global serum lipidomic profiles and evaluate whether the changes in inflammatory variables (CRP, TNF-α and IL-6) are reflected in the global lipidomic profiles of healthy adults.
METHODS: We performed UPLC/MS-based global lipidomic platform analysis of serum samples (n = 26) in a substudy of a randomised, double-blind, placebo-controlled 3-wk clinical intervention trial investigating the immunomodulatory effects of probiotics in healthy adults.
RESULTS: A total of 407 lipids were identified, corresponding to 13 different lipid classes. Serum samples showed decreases in the levels of lysophosphatidylcholines (LysoGPCho), sphingomyelins (SM) and several glycerophosphatidylcholines (GPCho), while triacylglycerols (TAG) were mainly increased in the probiotic LGG group during the intervention. Among the inflammatory variables, IL-6 was moderately associated by changes in global lipidomic profiles, with the top-ranked lipid associated with IL-6 being the proinflammatory LysoGPCho (20:4). There was a weak association between the lipidomic profiles and the two other inflammatory markers, TNF-α and CRP.
CONCLUSION: This was the first study to investigate the effects of probiotic intervention on global lipidomic profiles in humans. There are indications that probiotic LGG intervention may lead to changes in serum global lipid profiles, as reflected in decreased GPCho, LysoGPCho and SM as well as mainly increased TAG.
doi:10.3748/wjg.14.3188
PMCID: PMC2712851  PMID: 18506924
Probiotic; Lactobacillus rhamnosus GG; Lipidomic; Inflammatory mediators; Healthy adults
19.  Hexadecenoic Fatty Acid Isomers in Human Blood Lipids and Their Relevance for the Interpretation of Lipidomic Profiles 
PLoS ONE  2016;11(4):e0152378.
Monounsaturated fatty acids (MUFA) are emerging health biomarkers, and in particular the ratio between palmitoleic acid (9cis-16:1) and palmitic acid (16:0) affords the delta-9 desaturase index that is increased in obesity. Recently, other positional and geometrical MUFA isomers belonging to the hexadecenoic family (C16 MUFA) were found in circulating lipids, such as sapienic acid (6cis-16:1), palmitelaidic acid (9trans-16:1) and 6trans-16:1. In this work we report: i) the identification of sapienic acid as component of human erythrocyte membrane phospholipids with significant increase in morbidly obese patients (n = 50) compared with age-matched lean controls (n = 50); and ii) the first comparison of erythrocyte membrane phospholipids (PL) and plasma cholesteryl esters (CE) in morbidly obese patients highlighting that some of their fatty acid levels have opposite trends: increases of both palmitic and sapienic acids with the decrease of linoleic acid (9cis,12cis-18:2, omega-6) in red blood cell (RBC) membrane PL were reversed in plasma CE, whereas the increase of palmitoleic acid was similar in both lipid species. Consequentially, desaturase enzymatic indexes gave different results, depending on the lipid class used for the fatty acid content. The fatty acid profile of morbidly obese subjects also showed significant increases of stearic acid (C18:0) and C20 omega-6, as well as decreases of oleic acid (9cis-18:1) and docosahexaenoic acid (C22:6 omega-3) as compared with lean healthy controls. Trans monounsaturated and polyunsaturated fatty acids were also measured and found significantly increased in both lipid classes of morbidly obese subjects. These results highlight the C16 MUFA isomers as emerging metabolic marker provided that the assignment of the double bond position and geometry is correctly performed, thus identifying the corresponding lipidomic pathway. Since RBC membrane PL and plasma CE have different fatty acid trends, caution must also be used in the choice of lipid species for the interpretation of lipidomic profiles.
doi:10.1371/journal.pone.0152378
PMCID: PMC4821613  PMID: 27045677
20.  Proteomic and lipidomic signatures of lipid metabolism in NASH-associated hepatocellular carcinoma 
Cancer research  2013;73(15):10.1158/0008-5472.CAN-12-3797.
Non-alcoholic steatohepatitis (NASH) is a common pre-neoplastic condition of hepatocellular carcinoma (HCC). Mice with hepatocytic deletion of Pten develop NASH and HCC later in life. This model is highly valuable for studies aimed at identifying the molecular mechanism by which metabolic disorders contribute to tumor development. We applied proteomic and lipidomic profiling approaches to Pten null NASH liver and tumors. Circulating fatty acid composition was also characterized in these mice. The relevance to human NASH and HCC was further validated. This integrative proteomic and lipidomic study from mouse to human and from liver to blood, identified the following disease signatures: 1- an HCC signature: upregulated hepatic scd1/scd2, fads2 and acsl5:acsl1 ratio, elevated vaccenic and erucic acids and reduced margaric and linoleic acids in both liver and plasma; 2- a NASH signature that correlates with tumor burden: upregulated hepatic elovl6, elevated oleic, adrenic and osbond acids and reduced cervonic acid in liver and plasma; and 3- a NASH signature: reduced hepatic and circulating lignoceric and eicosapentaenoic acids. Altogether, these results demonstrate the role of lipid-modifying enzymes converting SFAs to MUFAs in HCC and the importance of an increased ratio of long chain n6-PUFAs over n3-PUFAs in NASH and HCC risk. They also highlight the relevance of the Pten null model for studies related to NASH and HCC and demonstrate that circulating lipid metabolome provides a direct read of lipid changes in the liver. Most importantly, novel candidate targets for HCC diagnosis, therapy, risk assessment and prevention were identified.
doi:10.1158/0008-5472.CAN-12-3797
PMCID: PMC3855016  PMID: 23749645
Liver cancer; proteomics; lipidomics; NASH
21.  An automated shotgun lipidomics platform for high throughput, comprehensive, and quantitative analysis of blood plasma intact lipids 
Blood plasma has gained protagonism in lipidomics studies due to its availability, uncomplicated collection and preparation, and informative readout of physiological status. At the same time, it is also technically challenging to analyze due to its complex lipid composition affected by many factors, which can hamper the throughput and/or lipidomics coverage. To tackle these issues, we developed a comprehensive, high throughput, and quantitative mass spectrometry-based shotgun lipidomics platform for blood plasma lipid analyses. The main hallmarks of this technology are (i) it is comprehensive, covering 22 quantifiable different lipid classes encompassing more than 200 lipid species; (ii) it is amenable to high-throughput, with less than 5 min acquisition time allowing the complete analysis of 200 plasma samples per day; (iii) it achieves absolute quantification, by inclusion of internal standards for every lipid class measured; (iv) it is highly reproducible, achieving an average coefficient of variation of <10% (intra-day), approx. 10% (inter-day), and approx. 15% (inter-site) for most lipid species; (v) it is easily transferable allowing the direct comparison of data acquired in different sites. Moreover, we thoroughly assessed the influence of blood stabilization with different anticoagulants and freeze-thaw cycles to exclude artifacts generated by sample preparation.
Practical applications: This shotgun lipidomics platform can be implemented in different laboratories without compromising reproducibility, allowing multi-site studies and inter-laboratory comparisons. This possibility combined with the high-throughput, broad lipidomic coverage and absolute quantification are important aspects for clinical applications and biomarker research.
doi:10.1002/ejlt.201500145
PMCID: PMC4606567  PMID: 26494980
Blood plasma; Clinical biomarker; High throughput; Lipids; Shotgun lipidomics
22.  Neuronal human BACE1 knockin induces systemic diabetes in mice 
Diabetologia  2016;59:1513-1523.
Aims
β-Secretase 1 (BACE1) is a key enzyme in Alzheimer’s disease pathogenesis that catalyses the amyloidogenic cleavage of amyloid precursor protein (APP). Recently, global Bace1 deletion was shown to protect against diet-induced obesity and diabetes, suggesting that BACE1 is a potential regulator of glucose homeostasis. Here, we investigated whether increased neuronal BACE1 is sufficient to alter systemic glucose metabolism, using a neuron-specific human BACE1 knockin mouse model (PLB4).
Methods
Glucose homeostasis and adiposity were determined by glucose tolerance tests and EchoMRI, lipid species were measured by quantitative lipidomics, and biochemical and molecular alterations were assessed by western blotting, quantitative PCR and ELISAs. Glucose uptake in the brain and upper body was measured via 18FDG-PET imaging.
Results
Physiological and molecular analyses demonstrated that centrally expressed human BACE1 induced systemic glucose intolerance in mice from 4 months of age onward, alongside a fatty liver phenotype and impaired hepatic glycogen storage. This diabetic phenotype was associated with hypothalamic pathology, i.e. deregulation of the melanocortin system, and advanced endoplasmic reticulum (ER) stress indicated by elevated central C/EBP homologous protein (CHOP) signalling and hyperphosphorylation of its regulator eukaryotic translation initiation factor 2α (eIF2α). In vivo 18FDG-PET imaging further confirmed brain glucose hypometabolism in these mice; this corresponded with altered neuronal insulin-related signalling, enhanced protein tyrosine phosphatase 1B (PTP1B) and retinol-binding protein 4 (RBP4) levels, along with upregulation of the ribosomal protein and lipid translation machinery. Increased forebrain and plasma lipid accumulation (i.e. ceramides, triacylglycerols, phospholipids) was identified via lipidomics analysis.
Conclusions/interpretation
Our data reveal that neuronal BACE1 is a key regulator of metabolic homeostasis and provide a potential mechanism for the high prevalence of metabolic disturbance in Alzheimer’s disease.
Electronic supplementary material
The online version of this article (doi:10.1007/s00125-016-3960-1) contains peer-reviewed but unedited supplementary material, which is available to authorised users.
doi:10.1007/s00125-016-3960-1
PMCID: PMC4901117  PMID: 27138913
18FDG-PET; Ceramide; ER stress; Glucose homeostasis; Insulin signalling; Neuronal BACE1
23.  Toward an Animal Model of the Human Tear Film: Biochemical Comparison of the Mouse, Canine, Rabbit, and Human Meibomian Lipidomes 
Purpose.
Secretions that are produced by meibomian glands (also known as meibum) are a major source of lipids for the ocular surface of humans and animals alike. Many animal species have been evaluated for their meibomian lipidomes. However, there have been a very small number of studies in which the animals were compared with humans side by side. Therefore, the purpose of this study was to compare meibum collected from humans and three typical laboratory animals, canines, mice, and rabbits, for their meibomian lipid composition in order to determine which animal species most resembles humans.
Methods.
High pressure liquid chromatography (HPLC) and gas-liquid chromatography (GLC) in combination with mass spectrometry were used to evaluate lipidomes of all tested species.
Results.
Among three tested animal species, mice were found to be the closest match to humans in terms of their meibomian lipidomes, while canines were the second closest species. The lipids of these three species were close to each other structurally and, for most lipid classes, quantitatively. The rabbit meibomian lipidome, on the other hand, was vastly different from lipidomes of all other tested species. Interestingly, a previously described class of lipids, acylated omega-hydroxy fatty acids (OAHFA), was found to be present in every tested species as the major amphiphilic component of meibum.
Conclusions.
Our side by side comparison of the rabbit and the human meibum demonstrated their vast differences. Thus, the rabbit seems to be a poor animal model of the human tear film, at least when studying its biochemistry and biophysics.
A side by side comparison of the rabbit and the human meibum demonstrated their vast biochemical differences. Thus, the rabbit seems to be a poor animal model of the human tear film studies. Mice and canines, on the other hand, were found to be very similar to humans and should be considered instead.
doi:10.1167/iovs.12-10516
PMCID: PMC3466071  PMID: 22918629
24.  Lipidomic Analysis of the Retina in a Rat Model of Smith-Lemli-Opitz Syndrome: Alterations in Docosahexaenoic Acid Content of Phospholipid Molecular Species 
Journal of neurochemistry  2007;105(3):1032-1047.
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 to the human disease. We evaluated alterations in the non-sterol lipid components of the retina in this model, compared to 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 two or three 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 molecular species in control retinas were 18:0–22:6 and di-22:6; notably, AY9944 treatment resulted in >80% less di-22:6 phosphatidylserine, 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 phosphatidylserine 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.
doi:10.1111/j.1471-4159.2007.05203.x
PMCID: PMC2721972  PMID: 18182048
Docosahexaenoic acid; fatty acid; lipidomic analysis; retina; AY9944
25.  Lactosomes: structural and compositional classification of unique nanometer-sized protein lipid particles of human milk 
Milk fat globules (MFG) are accepted primarily as triacylglycerol delivery systems. The identification of nanometer-sized lipid-protein particles termed ‘lactosomes’ that do not contain triacylglycerol raises the question of their possible functions. MFGs were isolated by slow centrifugation and lactosomes were isolated by ultracentrifugation at a density equivalent to plasma HDL (d > 1.063 g/ml) from human milk obtained from six volunteers at different lactation stages. Isolated lactosomes were analyzed and compared with MFGs for their size distribution, lipidome, proteome, and functional activity. Lactosomes from early milk—day 8, were found to be similar in size as those from mature milk >28 days averaging ~25 nm in diameter. In total, 97 non-redundant proteins were identified in the MFG and lactosome fractions, 46 of which were unique to the MFG fraction and 29 of which were unique to the lactosome fraction. The proteins identified in the lactosome and MFG fractions were enriched with proteins identified with immunomodulatory pathways. Unlike MFGs and GM1 laden reconstituted high- density lipoprotein (rHDL) that served as a positive control, lactosomal binding capacity to cholera toxin was weak. Lipidomic analyses found that lactosomes were devoid of triacylglycerol and gangliosides, unlike MFGs, but rich in a variety of phospholipid species. The data found differences in structure, composition and function between lactosomes and MFG suggesting these two particles are derived from different biosynthetic and/or secretory pathways. The results reveal a bioactive lipid-protein, nanometer-length scale particle that is secreted into milk not to supply energy to the infant, but to play unique, protective and regulatory roles.
doi:10.1021/jf102495s
PMCID: PMC3039697  PMID: 20925428
Milk; lipidome; proteome; fat globule; lactosome

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