Intestinal lipid alterations occur prior to antibody-induced prostaglandin E2 production in a mouse model of ischemia/reperfusion

Ischemia/reperfusion (IR) induced injury results in significant tissue damage in wild-type, but not antibody-deficient, Rag-1^−/− mice. However, Rag-1^−/− mice sustain intestinal damage after administration of wild-type antibodies or naturally occurring, specific anti-phospholipid related monoclonal antibodies, suggesting involvement of a lipid antigen. We hypothesized that IR initiates metabolism of cellular lipids, resulting in production of an antigen recognized by anti-phospholipid antibodies. At multiple time points after Sham or IR treatment, lipids extracted from mouse jejunal sections were analyzed by electrospray ionization triple quadrupole mass spectrometry. Within 15 min of reperfusion, IR induced significantly more lysophosphatidylcholine (lysoPC), lysophosphatidylglycerol (lysoPG) and free arachidonic acid (AA) production than Sham treatment. While lysoPC, lysoPG, and free AA levels were similar in C57Bl/6 (wild-type) and Rag-1^−/− mice, IR led to Cox-2 activation and prostaglandin E₂ (PGE₂) production in wild-type, but not in the antibody-deficient, Rag-1^−/− mice. Administration of wild-type antibodies to Rag-1^−/− mice restored PGE₂ production and intestinal damage. These data indicate that IR-induced intestinal damage requires antibodies for Cox-2 stimulated PGE₂ production but not for production of lysoPC and free AA.     

Lipidomics of host-pathogen interactions

The cell biology of intracellular pathogens (viruses, bacteria, eukaryotic parasites) has provided us with molecular information of host-pathogen interactions. As a result it is becoming increasingly evident that lipids play important roles at various stages of host-pathogen interactions. They act in first line recognition and host cell signaling during pathogen docking, invasion and intracellular trafficking. Lipid metabolism is a housekeeping function in energy homeostasis and biomembrane synthesis during pathogen replication and persistence. Lipids of enormous chemical diversity play roles as immunomodulatory factors. Thus, novel biochemical analytics in combination with cell and molecular biology are a promising recipe for dissecting the roles of lipids in host-pathogen interactions.     

High-throughput phospholipid quantitation in mammalian cells using matrix-assisted laser desorption ionization-time of flight mass spectrometry with N-trifluoroacetyl-phosphatidylethanolamine as internal standard

A high-throughput method is described for quantitative analysis of phospholipids. The method comprises extraction of lipids, addition of the internal standard N-trifluoroacetyl-phosphatidylethanolamine, and final analysis using matrix-assisted laser desorption ionization mass spectrometry. Quantitative data are obtained by calibration directly in the sample matrix. Calibration with one phosphatidylcholine was found sufficient for quantification of all major phosphatidylcholines tested. The method is very sensitive, has broad application, and is easily applicable to any biological sample. The detection limit for phosphatidylcholines was clearly below 2 μg on the spot, requiring less than 4000 cells corresponding to about 1.6 μg cell dry mass.     

磷脂分析方法的研究进展

磷脂是细胞膜的重要结构组成成分,是许多生理活性物质的前体物质,还发挥着细胞信号传导,细胞增殖,细胞凋亡等作用。研究表明许多疾病与磷脂的代谢异常有关,对磷脂的分析有助于疾病的诊断和治疗,对磷脂的研究已经成为医学,生物和药学等众多学科的研究热点,因为磷脂结构复杂,种类繁多,对磷脂的分析一直比较困难,随着分析技术的不断进步,特别是质谱技术,文献报道应用HPLC-ESI-MS进行磷脂分析的报道也越来越多,为了更好地认识磷脂的结构,功能及其代谢过程等,本文从磷脂的提取方法,色谱分析,质谱检测方法和代谢组学研究等四个方面对对近年来针对磷脂分析的研究进展加以综述。     

Surface analysis of lipids by mass spectrometry: More than just imaging

Mass spectrometry is now an indispensable tool for lipid analysis and is arguably the driving force in the renaissance of lipid research. In its various forms, mass spectrometry is uniquely capable of resolving the extensive compositional and structural diversity of lipids in biological systems. Furthermore, it provides the ability to accurately quantify molecular-level changes in lipid populations associated with changes in metabolism and environment; bringing lipid science to the “omics” age. The recent explosion of mass spectrometry-based surface analysis techniques is fuelling further expansion of the lipidomics field. This is evidenced by the numerous papers published on the subject of mass spectrometric imaging of lipids in recent years. While imaging mass spectrometry provides new and exciting possibilities, it is but one of the many opportunities direct surface analysis offers the lipid researcher. In this review we describe the current state-of-the-art in the direct surface analysis of lipids with a focus on tissue sections, intact cells and thin-layer chromatography substrates. The suitability of these different approaches towards analysis of the major lipid classes along with their current and potential applications in the field of lipid analysis are evaluated.     

Mass spectrometry imaging: Towards a lipid microscope?

Biological imaging techniques are the most efficient way to locally measure the variation of different parameters on tissue sections. These analyses are gaining increasing interest since 20 years and allow observing extremely complex biological phenomena at lower and lower time and resolution scale. Nevertheless, most of them only target very few compounds of interest, which are chosen a priori, due to their low resolution power and sensitivity. New chemical imaging technique has to be introduced in order to overcome these limitations, leading to more informative and sensitive analyses for biologists and physicians.Two major mass spectrometry methods can be efficiently used to generate the distribution of biological compounds over a tissue section. Matrix-Assisted Laser Desorption/Ionisation-Mass Spectrometry (MALDI-MS) needs the co-crystallization of the sample with a matrix before to be irradiated by a laser, whereas the analyte is directly desorbed by a primary ion bombardment for Secondary Ion Mass Spectrometry (SIMS) experiments. In both cases, energy used for desorption/ionization is locally deposited -some tens of microns for the laser and some hundreds of nanometers for the ion beam- meaning that small areas over the surface sample can be separately analyzed. Step by step analysis allows spectrum acquisitions over the tissue sections and the data are treated by modern informatics software in order to create ion density maps, i.e., the intensity plot of one specific ion versus the (x,y) position.Main advantages of SIMS and MALDI compared to other chemical imaging techniques lie in the simultaneous acquisition of a large number of biological compounds in mixture with an excellent sensitivity obtained by Time-of-Flight (ToF) mass analyzer. Moreover, data treatment is done a posteriori, due to the fact that no compound is selectively marked, and let us access to the localization of different lipid classes in only one complete acquisition.     

Analysis of lipids from crude lung tissue extracts by desorption electrospray ionization mass spectrometry and pattern recognition

A method is described using desorption electrospray ionization (DESI) mass spectrometry (MS) to obtain phospholipid mass spectral profiles from crude lung tissue extracts. The measured DESI mass spectral lipid fingerprints were then analyzed by unsupervised learning principal components analysis (PCA). This combined approach was used to differentiate the effect(s) of two vaccination routes on lipid composition in mouse lungs. Specifically, the two vaccination routes compared were intranasal (i.n.) and intradermal (i.d.) inoculation of the Francisella tularensis live vaccine strain (Ft–LVS). Lung samples of control and LVS-inoculated mice were quickly extracted with a methanol/chloroform solution, and the crude extract was directly analyzed by DESI–MS, with a total turnaround time of less than 10 min/sample. All of the measured DESI mass spectra (in both positive and negative ion mode) were compared via PCA, resulting in clear differentiation of mass spectral profiles of i.n.-inoculated mouse lung tissues from those of i.d.-inoculated and control mouse lung tissues. Lipid biomarkers responsible for sample differentiation were identified via tandem MS (MS/MS) measurements or by comparison with mass spectra of lipid standards. The DESI–MS approach described here provided a practical and rapid means to analyze tissue samples without extensive extractions and solvent changes.     

Comparative lipidomic analysis of mouse and human brain with Alzheimer disease

Lipids are key regulators of brain function and have been increasingly implicated in neurodegenerative disorders including Alzheimer disease (AD). Here, a systems-based approach was employed to determine the lipidome of brain tissues affected by AD. Specifically, we used liquid chromatography-mass spectrometry to profile extracts from the prefrontal cortex, entorhinal cortex, and cerebellum of late-onset AD (LOAD) patients, as well as the forebrain of three transgenic familial AD (FAD) mouse models. Although the cerebellum lacked major alterations in lipid composition, we found an elevation of a signaling pool of diacylglycerol as well as sphingolipids in the prefrontal cortex of AD patients. Furthermore, the diseased entorhinal cortex showed specific enrichment of lysobisphosphatidic acid, sphingomyelin, the ganglioside GM3, and cholesterol esters, all of which suggest common pathogenic mechanisms associated with endolysosomal storage disorders. Importantly, a significant increase in cholesterol esters and GM3 was recapitulated in the transgenic FAD models, suggesting that these mice are relevant tools to study aberrant lipid metabolism of endolysosomal dysfunction associated with AD. Finally, genetic ablation of phospholipase D(2), which rescues the synaptic and behavioral deficits of an FAD mouse model, fully normalizes GM3 levels. These data thus unmask a cross-talk between the metabolism of phosphatidic acid, the product of phospholipase D(2), and gangliosides, and point to a central role of ganglioside anomalies in AD pathogenesis. Overall, our study highlights the hypothesis generating potential of lipidomics and identifies novel region-specific lipid anomalies potentially linked to AD pathogenesis.     

Targeted eicosanoids lipidomics of exhaled breath condensate in healthy subjects

Background

Exhaled breath condensate collection is a non-invasive method of sampling the respiratory tract that can be repeated several times in a wide range of clinical settings. Quantitation of non-volatile compounds in the condensate requires highly sensitive analytical methods, e.g. mass spectrometry.

Objective

To validate cross-platform measurements of eicosanoids using high performance liquid chromatography or gas chromatography coupled with mass spectrometry in exhaled breath condensate sampled from 58 healthy individuals.

Methods

Twenty different eicosanoid compounds, representing major arachidonic acid lipoxygenation and cyclooxygenation pathways were measured using a stable isotope dilution method. We applied a free palmitic acid concentration as a surrogate marker for the condensate dilution factor.

Results

Eicosanoids concentrations in the condensates were consistent with their content in other biological fluids. Prostaglandin E₂ was the most abundant mediator, represented by its stable metabolite tetranor-PGEM. Prostaglandin D₂ products were at low concentration, while hydroxyacids derived from lipoxygenation were abundant. 5-HETE was elevated in current tobacco smokers. Leukotriene B₄ has the highest concentration of all 5-LO products. 15-LO analogues of cysteinyl leukotrienes–eoxins were detectable and metabolized to eoxin E₄. Two main vascular prostanoids: prostacyclin and thromboxane B₂ were present as metabolites. A marker for non-enzymatic lipid peroxidation, 8-iso-PGF_2α isoprostane was increased in smokers.

Conclusion

Presented targeted lipidomics analysis of exhaled breath condensate in healthy subjects justifies its application to investigation of inflammatory lung diseases. Measurements of non-volatile mediators of inflammation in the condensates might characterize disease-specific pathological mechanisms and responses to treatment.     

Investigation of the molecular ion structure for aldononitrile acetate derivatized muramic acid

The muramic acid assay is a powerful tool for detecting both intact bacteria and bacterial debris. Past use of aldononitrile acetate derivatization for determining muramic acid in complex samples by gas chromatography/mass spectrometry met detection needs in many instances; however, questions have been raised regarding the interpretation of the derivative structure and its electron ionization fragments. In this study, we applied different methods and proved that the aldononitrile acetate derivatized muramic acid yields a molecular weight of 398, associated with a lactam structure. We also presented evidence that the structure of aldononitrile acetate derivatized muramic acid is acetylated at four positions, 3 O-acetylations and 1N-acetylation. In practical manner, this communication provides a comprehensive reference to researchers using δ¹³C value or ion fragments of the muramic acid marker in biogeochemical studies.     

Pathway-oriented profiling of lipid mediators in macrophages

Macrophages produce various kinds of lipid mediators including eicosanoids and platelet-activating factor. Since they are produced from common precursors, arachidonic acid-containing phospholipids, regulations of metabolic pathways underlie the patterning of lipid mediator production. Here, we report a pathway-oriented profiling strategy of lipid mediators by a newly developed multiplex quantification system. We profiled mouse peritoneal macrophages in different activation states. The analysis of kinetics revealed the differences in the production time course of various lipid mediators, which also differed by the macrophage types. Scatterplot matrix analysis of the inhibitor study revealed correlations of lipid mediator species. The changes of these correlations provided estimates on the effects of lipopolysaccharide priming. We also found a highly linked production of 11-hydroxyeicosatetraenoic acid and prostaglandin E2, implying the in vivo property of cyclooxygenase-mediated 11-hydroxyeicosatetraenoic acid production. The present approach will serve as a strategy for understanding the regulatory mechanism of lipid mediator production.     

Imaging mass spectrometry of thin tissue sections: a decade of collective efforts

Imaging mass spectrometry (MS) allows to monitor the spatial distribution and abundance of endogenous and administered compounds present within tissue specimens. Several different but complementary imaging MS technologies have been developed allowing the analysis of a wide variety of compounds including inorganic elementals, metabolites, lipids, peptides, proteins and xenobiotics with spatial resolutions from micrometer to nanometer scales. In the past decade, an enormous collective body of work has been done to develop and improve the imaging MS technology. This article gives a historical perspective, an overview of the principle and status of the technology and lists the main fields of applications. It also enumerates some of the critical challenges we need to collectively address for imaging MS to be considered a mainstream analytical method.     

Shotgun cholanomics of ileal fluid

In this study we have developed a rapid method for the shotgun analysis of bile acids in intestinal fluid. The method is semi-quantitative, and requires little sample preparation. Bile salts might contribute to the pathogenesis of Crohn's disease. In a pilot study we demonstrate the method by analysing the bile acid content of ileal fluid from seven Crohn's disease patients and three healthy controls. The dominant bile acids observed were di and/or trihydroxycholanoates, di- and/or trihydroxycholanoylglycines, di- and/or tri-hydroxycholanoyltaurines, monosulphated dihydroxycholanoates and monosulphated dihydroxycholanoylglycine. The method can be similarly applied to samples derived from other parts of the intestine.     

Diurnal Regulation of Lipid Metabolism and Applications of Circadian Lipidomics

The circadian timing system plays a key role in orchestrating lipid metabolism. In concert with the solar cycle, the circadian system ensures that daily rhythms in lipid absorption, storage, and transport are temporally coordinated with rest-activity and feeding cycles. At the cellular level, genes involved in lipid synthesis and fatty acid oxidation are rhythmically activated and repressed by core clock proteins in a tissue-specific manner. Consequently, loss of clock gene function or misalignment of circadian rhythms with feeding cycles （e.g., in shift work） results in impaired lipid homeostasis. Herein, we review recent progress in circadian rhythms research using lipidomics, i.e., large-scale profiling of lipid metabolites, to characterize circadian-regulated lipid pathways in mammals. In mice, novel regulatory circuits involved in fatty acid metabolism have been identified in adipose tissue, liver, and muscle. Extensive diversity in circadian regulation of plasma lipids has also been revealed in humans using lipidomics and other metabolomics approaches. In future studies, lipidomics platforms will be increasingly used to better understand the effects of genetic variation, shift work, food intake, and drugs on circadian-regulated lipid pathways and metabolic health.