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

Concordant inter-laboratory derived concentrations of ceramides in human plasma reference materials via authentic standards

In this community effort, we compare measurements between 34 laboratories from 19 countries, utilizing mixtures of labelled authentic synthetic standards, to quantify by mass spectrometry four clinically used ceramide species in the NIST (National Institute of Standards and Technology) human blood plasma Standard Reference Material (SRM) 1950, as well as a set of candidate plasma reference materials (RM 8231). Participants either utilized a provided validated method and/or their method of choice. Mean concentration values, and intra- and inter-laboratory coefficients of variation (CV) were calculated using single-point and multi-point calibrations, respectively. These results are the most precise (intra-laboratory CVs ≤ 4.2%) and concordant (inter-laboratory CVs < 14%) community-derived absolute concentration values reported to date for four clinically used ceramides in the commonly analyzed SRM 1950. We demonstrate that calibration using authentic labelled standards dramatically reduces data variability. Furthermore, we show how the use of shared RM can correct systematic quantitative biases and help in harmonizing lipidomics. Collectively, the results from the present study provide a significant knowledge base for translation of lipidomic technologies to future clinical applications that might require the determination of reference intervals (RIs) in various human populations or might need to estimate reference change values (RCV), when analytical variability is a key factor for recall during multiple testing of individuals

Concordant inter-laboratory derived concentrations of ceramides in human plasma reference materials via authentic standards

In this community effort, we compare measurements between 34 laboratories from 19 countries, utilizing mixtures of labelled authentic synthetic standards, to quantify by mass spectrometry four clinically used ceramide species in the NIST (National Institute of Standards and Technology) human blood plasma Standard Reference Material (SRM) 1950, as well as a set of candidate plasma reference materials (RM 8231). Participants either utilized a provided validated method and/or their method of choice. Mean concentration values, and intra- and inter-laboratory coefficients of variation (CV) were calculated using single-point and multi-point calibrations, respectively. These results are the most precise (intra-laboratory CVs ≤ 4.2%) and concordant (inter-laboratory CVs < 14%) community-derived absolute concentration values reported to date for four clinically used ceramides in the commonly analyzed SRM 1950. We demonstrate that calibration using authentic labelled standards dramatically reduces data variability. Furthermore, we show how the use of shared RM can correct systematic quantitative biases and help in harmonizing lipidomics. Collectively, the results from the present study provide a significant knowledge base for translation of lipidomic technologies to future clinical applications that might require the determination of reference intervals (RIs) in various human populations or might need to estimate reference change values (RCV), when analytical variability is a key factor for recall during multiple testing of individuals

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

Highlighting anatomical sub-structures in rat brain tissue using lipid imaging

Cell membranes are made up of a mixture of glycerolipids, sphingolipids, gangliosides and cholesterol. Lipids play important roles in a cell’s life. However many of their functions have still to be discovered. In the present work, we describe an efficient, easy and rapid methodology to accurately localize phosphatidylcholines and sphingomyelins from a single coronal rat brain section in the cerebrum area. Matrix assisted laser desorption/ionization (MALDI) mass spectrometry was used to profile and image lipids. The best resolved structure was 25–50 μm in the hippocampus

Enhancing structural elucidation of the lipidome through optimized SFC-HRMS/MS hyphenation

International audienceSFC separation on packed column for lipids analysis is well adapted thanks to the interclass lipid species separation as normal phase liquid chromatography1. The characterization of the lipidome typically requires the use of Data Dependent Acquisition (DDA) MS/MS method, which is limited to the fragmentation to the most intense signals during a cycle time2. Considering this issue, the strategy involving the iterative ion exclusion consisted of automatically placing prior fragmented ions on a list along sequential repetitive injections seems well adapted3. However, some MS/MS spectra at lower intensity present fragment ions of interest with weak signal-to-noise ratio. In this context, MS/MS acquisition method parameters are critical to ensure a short cycle time that meets this minimum requirements for an optimal coverage of lipid species in complex biological matrices. SFC gradient and MS/MS parameters of the Q/TOF were optimized to address this issue. A mix of lipid standards representing of 27 lipid classes were injected to determine the optimized parameters for the highly confident structural elucidation of lipid species present in complex biological matrice

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