Mitochondrial dysfunction governs immunometabolism in leukocytes of patients with acute-on-chronic liver failure.

Background & aims: Patients with acute-on-chronic liver failure (ACLF) present a systemic hyperinflammatory response associated with increased circulating levels of small-molecule metabolites. To investigate whether these alterations reflect inadequate cell energy output, we assessed mitochondrial morphology and central metabolic pathways with emphasis on the tricarboxylic acid (TCA) cycle in peripheral leukocytes from patients with acutely decompensated (AD) cirrhosis, with and without ACLF. Methods: The study included samples from patients with AD cirrhosis (108 without and 128 with ACLF) and 41 healthy individuals. Leukocyte mitochondrial ultrastructure was visualized by transmission electron microscopy and cytosolic and mitochondrial metabolic fluxes were determined by assessing NADH/FADH2 production from various substrates. Plasma GDF15 and FGF21 were determined by Luminex and acylcarnitines by LC-MS/MS. Gene expression was analyzed by RNA-sequencing and PCR-based glucose metabolism profiler array. Results: Mitochondrial ultrastructure in patients with advanced cirrhosis was distinguished by cristae rarefication and swelling. The number of mitochondria per leukocyte was higher in patients, accompanied by a reduction in their size. Increased FGF21 and C6:0- and C8:0-carnitine predicted mortality whereas GDF15 strongly correlated with a gene set signature related to leukocyte activation. Metabolic flux analyses revealed increased energy production in mononuclear leukocytes from patients with preferential involvement of extra-mitochondrial pathways, supported by upregulated expression of genes encoding enzymes of the glycolytic and pentose phosphate pathways. In patients with ACLF, mitochondrial function analysis uncovered break-points in the TCA cycle at the isocitrate dehydrogenase and succinate dehydrogenase level, which were bridged by anaplerotic reactions involving glutaminolysis and nucleoside metabolism. Conclusions: Our findings provide evidence at the cellular, organelle and biochemical levels that severe mitochondrial dysfunction governs immunometabolism in leukocytes from patients with AD cirrhosis and ACLF. Lay summary: Patients at advanced stages of liver disease have dismal prognosis due to vital organ failures and the lack of treatment options. In this study, we report that the functioning of mitochondria, which are known as the cell powerhouse, is severely impaired in leukocytes of these patients, probably as a consequence of intense inflammation. Mitochondrial dysfunction is therefore a hallmark of advanced liver disease

Etudes structurales, biochimiques et moléculaires des Sulfogalactosylcéramides (application à la leucodystrophie métachromatique de l'adulte)

La leucodystrophie métachromatique est une maladie neurologique rare d origine génétique qui se décline en deux formes cliniques distinctes chez l adulte : les formes motrices qui se présentent principalement par une paraparésie spastique ou une ataxie cérébro-spinale, entraînant des troubles progressifs de la marche, et les formes psycho-cognitives qui se caractérisent par l apparition de troubles mentaux et de troubles du comportement de type schizophrénique dès le début de la maladie. Cette maladie prédominante du système nerveux central se caractérise par un trouble du métabolisme de la myéline, due à un déficit en arylsulfatase A, enzyme de dégradation des sulfogalactosylcéramides (sulfatides), sphingoglycolipides de la série gala . L étude effectuée en biologie moléculaire sur le gène de l arylsulfatase A a montré l existence d une relation génotype/phénotype par la présence de mutations spécifiques liées aux formes de l adulte. Les sulfatides s accumulent en très grande quantité dans les cellules et sont alors excrétés dans les urines des patients (sulfatidurie). Nous avons à travers ce travail, mis au point les techniques de détections des sulfatides urinaires par immunodétection sur couche mince à l aide d un anticorps anti-sulfatide OL-2, caractérisé au laboratoire, et quantifié ces molécules par spectrométrie de masse en tandem avec un sulfatide synthétique non physiologique. Les études spectrométriques ont permis de mettre en évidence une nouvelle base sphingoïde (la sphingadiénine d18:2) dans ces composés, elle est présente dans tous les sphingoglycolipides de la série gala . La présence de cette nouvelle base sphingoïde ouvre une nouvelle voie de recherche sur le métabolisme des sphingoglycolipides.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

The Lipid World Concept of Plant Lipidomics

Lipidomics has emerged as a new field that allows using various approaches to the chemical structures and the quantitative composition of more than a hundred lipid molecular species constituting the cellular lipidome. The increase of the performance of lipidomic analysis has resulted in recent developments in electrospray ionization mass spectrometry (ESI/MS) and rapid scanning tandem spectrometers that are capable of detecting and quantifying lipids at high sensitivity in an online high-performance chromatography. In this review, after a short description of the characteristic lipid classes of the plant kingdom, different approaches of 'lipidomics' will be addressed, including sample preparation (extractions, sample storage), MS analysis (ionization sources, shotgun lipidomics by direct infusion using tandem-in-space instruments and high-resolution systems and the use of separative methods before MS studies). Common fragmentation modes (MRM, CID including HCD) to determine molecular structures of lipid families in plants are also developed. The different principles of MS lipid analyses are briefly described and the different strategies using HPLC and MS/MS to quantify the different plant lipid molecular species are presented

Recent methodological developments in data-dependent analysis and data-independent analysis workflows for exhaustive lipidome coverage

International audienceUntargeted lipidomics applied to biological samples typically involves the coupling of separation methods to high-resolution mass spectrometry (HRMS). Getting an exhaustive coverage of the lipidome with a high confidence in structure identification is still highly challenging due to the wide concentration range of lipids in complex matrices and the presence of numerous isobaric and isomeric species. The development of innovative separation methods and HRMS(/MS) acquisition workflows helped improving the situation but issues still remain regarding confident structure characterization. To overcome these issues, thoroughly optimized MS/MS acquisition methods are needed. For this purpose, different methodologies have been developed to enable MS and MS/MS acquisition in parallel. Those methodologies, derived from the proteomics, are referred to Data Dependent Acquisition (DDA) and Data Independent Acquisition (DIA). In this context, this perspective paper presents the latest developments of DDA- and DIA-based lipidomic workflows and lists available bioinformatic tools for the analysis of resulting spectral data

Experimental Evidence That Electrospray-Produced Sodiated Lysophosphatidyl Ester Structures Exist Essentially as Protonated Salts

Sodiated lysoglycerophosphatidylethanolamine (LGPE) and lysoglycerophosphatidylcholine (LGPC) species dissociate under low collision energy by covalent bond cleavage resulting in product ions with either sodium retention or without sodium retention. For explaining these fragmentations, sodium chelation by heteroatoms (as charge-solvated structures) is often considered, and consequently, under keV collision conditions, sodium is "spectator" of cleavages (charge remote fragmentation). However, cleavage of such charge-solvated forms under low-energy conditions should result in sodium desolvation rather than covalent bond cleavage. In the present study, protonated salts are proposed as the main representative structures of the sodiated LGPE and LGPC forms. These structures are generated from sodiation of zwitterionic and betaine forms of LGPE and LGPC molecules, respectively. Experimental evidence to determine which structure is involved in the dissociations is provided, especially by comparing the dissociation of LGPL sodiated forms with that of sodiated polyethylene glycols. Energy-resolved mass spectrometry breakdown experiments were performed on a quadrupole time-of-flight instrument to demonstrate that both LGPE and LGPC sodiated forms exist as protonated salt structures. From such structures, proton migration by prototropy can result in different bond cleavages whereas the salt moiety remains spectator of these processes

proFIA: A data preprocessing workflow for Flow Injection Analysis coupled to High-Resolution Mass Spectrometry

International audienceMotivation: Flow Injection Analysis coupled to High-Resolution Mass Spectrometry (FIA-HRMS) is a promising approach for high-throughput metabolomics. FIA-HRMS data, however, cannot be preprocessed with current software tools which rely on liquid chromatography separation, or handle low resolution data only. Results: We thus developed the proFIA package, which implements a suite of innovative algorithms to preprocess FIA-HRMS raw files, and generates the table of peak intensities. The workflow consists of 3 steps: i) noise estimation, peak detection and quantification, ii) peak grouping across samples, and iii) missing value imputation. In addition, we have implemented a new indicator to quantify the potential alteration of the feature peak shape due to matrix effect. The preprocessing is fast (less than 15 s per file), and the value of the main parameters (ppm and dmz) can be easily inferred from the mass resolution of the instrument. Application to two metabolomics datasets (including spiked serum samples) showed high precision (96%) and recall (98%) compared with manual integration. These results demonstrate that proFIA achieves very efficient and robust detection and quantification of FIA-HRMS data, and opens new opportunities for high-throughput phenotyping. Availability: The proFIA software (as well as the plasFIA data set) is available as an R package on the Bioconductor repository (http://bioconductor.org/packages/proFIA), and as a Galaxy module on the Main Toolshed (https://toolshed.g2.bx.psu.edu/) and on the Workflow4Metabolomics online infrastructure (http://workflow4metabolomics.org). Contacts: [email protected] and [email protected]

Evaluation of the high-field orbitrap fusion for compound annotation in metabolomics

International audienceAnnotation of signals of interest represents a key point in mass spectrometry-based metabolomics studies. The first level of investigation is the elemental composition, which can be deduced from accurately measured masses and isotope patterns. However, accuracy of these two parameters remains to be evaluated on last generation mass spectrometers to determine the level of confidence that can be used during the annotation process. In this context, we evaluated the performance of the Orbitrap Fusion mass spectrometer for the first time and demonstrated huge potential for metabolite annotation via elemental composition determination. This work was performed using a set of 50 standard compounds analyzed under LC/MS conditions in solvent and biological media. Accurate control of the number of trapped ions proved mandatory to avoid space charge effects, ensure sub-ppm mass accuracy (using external calibration), and reliable measurement of isotopic patterns at 500,000 resolution. On the basis of the results, we propose standard optimized experimental conditions for performing robust and accurate untargeted metabolomics on the Orbitrap Fusion at high mass measurement and mass spectral accuracy

Separation of biologically relevant isomers on an Orbitrap mass spectrometer using high‐resolution drift tube ion mobility and varied drift gas mixtures

Rationale Atmospheric pressure drift tube ion mobility is a powerful addition to the Orbitrap mass spectrometer enabling direct separation of isomers. Apart from offering high resolving power in a compact design, it also facilitates optimization of the separation gas, as shown here for a series of biologically relevant isomer pairs.Methods An Excellims MA3100 High-Resolution Atmospheric Pressure Ion Mobility Spectrometer (HR-IMS) was coupled to a Thermo Scientific (TM) Q Exactive (TM) Focus hybrid quadrupole-Orbitrap (TM) mass spectrometer, using an Excellims Directspray (TM) Electrospray Ionization source and a gas mixture setup to provide various drift gases (air, CO2 and mixtures). This instrument combination was used to separate isomers of eight pairs of metabolites and gangliosides, optimizing drift gas conditions for best separation of each set.Results All but one of the isomers pairs provided could be partially or fully separated by the HR-IMS-MS combination using ion mobility drift times. About half of the separated compounds showed significantly better analytical separation when analyzed in a mixture of CO2 and air rather than air or CO2 alone. Resolving power of up to 102 was achieved using the 10 cm atmospheric drift tube ion mobility add-on for the Orbitrap mass spectrometer.Conclusions The present analysis demonstrates the usefulness of using atmospheric drift tube IMS on an Orbitrap mass spectrometer to characterize the isomeric composition of samples. It also highlights the potential benefits of being able to quickly optimize the drift gas composition to selectively maximize the mobility difference for isomer separation

A re-calibration procedure for interoperable lipid collision cross section values measured by traveling wave ion mobility spectrometry

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Baseline urine metabolic phenotype in patients with severe alcoholic hepatitis and its association with outcome

International audienceSevere alcoholic hepatitis (SAH) has a high mortality rate, and corticosteroid therapy is effective in 60% patients. This study aimed to investigate a baseline metabolic phenotype that could help stratify patients not likely to respond to steroid therapy and to have an unfavorable outcome. Baseline urine metabolome was studied in patients with SAH using ultra‐high performance liquid chromatography and high‐resolution mass spectrometry. Patients were categorized as responders (Rs, n = 52) and nonresponders (NRs, n = 8) at day 7 according to the Lille score. Multivariate projection analysis identified metabolites in the discovery cohort (n = 60) and assessed these in a validation cohort of 80 patients (60 Rs, 20 NRs). A total of 212 features were annotated by using metabolomic/biochemical/spectral databases for metabolite identification. After a stringent selection procedure, a total of nine urinary metabolites linked to mitochondrial functions significantly discriminated nonresponders, most importantly by increased acetyl‐L‐carnitine (12‐fold), octanoylcarnitine (4‐fold), decanoylcarnitine (4‐fold), and alpha‐ketoglutaric acid (2‐fold) levels. Additionally, urinary acetyl‐L‐carnitine and 3‐hydroxysebasic acid discriminated nonsurvivors ( P 0.3; P 3.0; P 2,500 ng/mL reliably segregated survivors from nonsurvivors ( P < 0.01, log‐rank test) in our study cohort. Conclusion: Urinary metabolome signatures related to mitochondrial functions can predict pretherapy steroid response and disease outcome in patients with SAH. ( Hepatology Communications 2018;2:628‐643