Lipidomic techniques and case studies, example of experimental designs focused on lipidomics applied to animal sciences
Laurence Bernard, Carole Delavaud, Hélène Fougère, Justine Bertrand-Michel INRA - VetAgro Sup, UMR 1213, Centre ARA, Saint-Genès-Champanelle, France MetaToul-LIPIDOMIQUE, I2MC, Inserm, Université Paul Sabatier UMR1048, Toulouse, France
NOM DE L’AUTEUR [email protected] JOUR / MOIS / ANNEE Will address the following... Lipids
What ? Definition and structure: molecular species
Where ? Location, biological functions
Why ? Interest in lipid determination
How? Techniques for lipid analysis => Lipidomics
Case study An application in dairy animals
2 MANNA 23/07/2019 Presentation . Definition and structure of Lipids . Lipidomics • Techniques of lipid analysis, • MS detection • Workflow (from sample preparation to analysis of the data); Approaches . Literature: example of applications . An example of application in dairy animals
.03 _01 Definition and structure of lipids: molecular species
.04 Lipid: Definition What?
No agreement in the lipid community:
• Lipids have been globally defined as biological substances that are generally hydrophobic in nature and in many cases soluble in organic solvents • Lipids are. fatty acids and their derivatives, and substances related biosynthetically or functionally to these compounds
• Lipids are hydrophobic or amphipathic small molecules that may originate entirely or in part by carbanion-based condensations of thioesters (fatty acids, polyketides, etc.) and/or by carbocation-based condensations of isoprene units (prenols, sterols, etc.)
=> These chemical properties cover a broad range of molecules, such as fatty acids, phospholipids, sterols, sphingolipids, terpenes, and others
Fahy, E. et al., 2005, Eur J Lipid Sci Tech • Christie et al., 2003 MANNA 23/07/2019 Lipids Where? • In various living organisms
• In all animal tissues and biological fluids
Why?
• Structural role (biological membrane)
• Store energy (AT Represents 10-50% in human, ~15% in bovine and 20% in porcine of the adult body weight; seeds...)
• Bioactive lipids (molecular signaling)
=> Key role in various diseases (metabolic, cancer, cardiovascular, inflammation...) and are impacted by nutrition
MANNA 23/07/2019 Lipids: molecular species in 8 categories
Consistent structure representation across categories (Lipid Maps Lipid Chemistry Tutorial) O FA
Fatty Acyls (FA) OH
O Glycerol Simple Glycerolipids (GL) O OH lipids O H (Triacylglycerides;TAG) O H
H Sterol Lipids (ST) H cholesterol O H H
O Phosphate O O P O Complex Glycerophospholipids (GP) O O + - N lipids (Phospholipids; PL) O H O O Sphingolipids (SP) H OH OH
NH H Amide O linkage
Prenol Lipids (PR) Saccharolipids (SL), Polyketides (PK)
MANNA 23/07/2019 An extensive range of quantities
Example of Lipid composition human plasma (from Christie et al, JLR, 1985)
(SP) Sphingolipid LPC
Cholesterol ester (ST) (PL) Phosphatidylcholine PC CE
(FA) Fatty acids Chol TAG Triacylglyceride (GL)
=> Modulated according to the species (Kaabia et al., 2018)
Kaabia et al. (2018) Plasma lipidomic analysis reveals strong similarities between lipid fingerprints in human, hamster and mouse compared to other animal species. Sci Rep 8, 9. MANNA 23/07/2019 An extensive range of quantities
Sphingolipids
Sphingomyelin
Sphingolipid (≈2%)
(Hammad SM et al. J. Lipid Res. 2010;51:3074-3087) MANNA 23/07/2019 An extensive range of molecules
Lipidomics reveals a remarkable diversity of lipids in human plasma:
(TAG,...) (PL)
Quehenberger et al. (2010) MANNA 23/07/2019 Structure of the fatty acids: = hydrocarbonated aliphatic chain; with a carboxylic function; 2 to 24 atoms of C; with or without double bond 16 2 1 CH3 1 • Saturated COOH 3
Palmitic acid – 16:0 6 COOH 18 CH3 9 Oleic acid – 18:1 cis9 (ω9) 18 6 CH3 • Ex of mono and COOH 9 12 polyunsaturated Linoleic acid – 18:2 cis9 cis12 (ω6) of C18
9 12 15 18 COOH 3 CH3 Linolenic acid – 18:3 cis9 cis12 cis15 (ω3) 11 18 Conjugated Linoleic CLA: COOH CH3 9 Acid (CLA) Rumenic acid – 18:2 cis9 trans11, c9t11-CLA • Branched chain fatty acids, hydroxyded fatty acids acids...
MANNA 23/07/2019 Structure of the fatty acids: Usual nomenclatures
Example of Linoleic acid n or
Δ CH3 18 Number of double bonds
Number of carbons 18:2 n-6 (or w6) or 18:2 Δ9,12 (cis9,cis12-18:2)
Position of the first double bond Position of double bonds from the methyl end from the carboxyl end
MANNA 23/07/2019 Oleic acid (18:1 cis9 or n-9) Cis configuration :
Adjacent hydrogen atoms are on the same side of the double bond.
The FA chain is curved Melting point: 13 °C
Trans configuration : (bacterial or technological origin) Elaidic acid (18:1 trans9, n-9) The two hydrogen atoms are bound to opposite sides of the double bond.
The FA chain is not curved Melting point: 44°C => Differences in geometry between the cis and trans MUFA play an important role in biological processes, in the construction of biological structures (such as cell membranes), confer technological properties (ex fat melting point)…
MANNA 23/07/2019 9
12 18 6 CH3 COOH 9 12 Linoleic acid – 18:2 cis9 cis12 (ω6)
Linoleic acid
c-9, c-12 isomer 6
Conjugated linoleic acid (CLA) 11 (in ruminant products)
28 possible configurations (isomers) numerous biological properties CLA c-9, t-11 isomer (cancers, obesity, antilipogenic…) (rumenic acid) 11 18 COOH CH3 9 Rumenic acid – 18:2 cis9 trans11, c9t11-CLA
MANNA 23/07/2019 Lipid MAPS Website: a huge source of information on lipids https://www.lipidmaps.org/
MANNA 23/07/2019 MANNA 23/07/2019 http://www.lipidmaps.org/resources/databases/index.php?tab=lmsd
Search based on structure....
MANNA 23/07/2019 LIPID MAPS Structure Database (LMSD) http://www.lipidmaps.org/data/structure/
Online functions: Browse Text search Substructure search Download structures Other Websites...
=> provide useful online resources for an overview of these molecules and their structures and methods for analysis
Lipid Library (http://www.lipidhome.co.uk/) by Christie Lipid Bank (http://www.lipidbank.jp) => The official database of Japanese Conference on the Biochemistry of Lipids Cyberlipids (http://www.cyberlipid.org) : This site for cyberlipid studies is an online, non-profit scientific organization whose purpose is to collect, study and diffuse information on all aspects of lipidology. 1.The site seeks to establish contacts between students, teachers, scientists and technicians and expose various models in all fields, forgotten studies of the past, work in progress and hot fields. 2.The site will try to feature an extensive, always upgraded, annotated bibliography devoted to the main presented topics.
MANNA 23/07/2019 _02
Lipidomics
.020 Omics Technologies: a field of study in Biology
Genomics
Transcriptomics Bioinformatics Proteomics
Metabolomics
Lipidomics
Is the part of metabolomics dedicated to lipids: the full molecular characterization of the lipid components in a defined system, as well as consideration of their function when available
MANNA 23/07/2019 Lipidomics: Definition and key points
• Lipidomics is the study of lipidomes (= a large variety of molecules with various pattern of concentration) • Allow a comprehensive analysis of lipid molecules ≈ 300 This term used in the titles of scientific articles: 1st in 2002, ≈ 300 the last 2 years
2002 2018 A discipline at the interface of lipid biology technology, biomedical science and animal science: Physiological or nutritional changes or pathology can induce changes in the profiles of endogenous lipids Is a powerful tool for the development of lipid biomarkers, lipid signatures to study disease states metabolism
(Lam and Shui, 2013 J Genet Genomics) (Fahy et al. 2005, EJLST) (Lagarde et al, 2003 BBA) (Liu et al, 2018 Progress in Lipid Research 71,70-85) MANNA 23/07/2019 Lipidomics: How?
Uses the techniques of analytical chemistry to study the entire lipid content of a cell, a tissue, a biological fluid...
Associated with mass spectrometry (MS): liquid- chromatography MS, shotgun lipidomics...
=> By techniques of lipid analysis + MS
=> An emerging discipline that evolved thanks to the increase in the analytical power of mass spectrometry (MS) and new developments in MS
MANNA 23/07/2019 Techniques of lipid Analysis
MANNA 23/07/2019 How? Main techniques for lipid analysis
Chromatography
• Analytical technique => separation of the constituents of a homogeneous liquid or gaseous phase mixture
• Principle based on the repartition of the compounds present in the mixture between two phases: the stationary phase (trapped in the column (GC, HPLC), on the plate (TLC)) and the mobile phase (gas or eluent) which moves
• Separation based on the intrinsic properties (size, structure, ...) of the different compounds and their affinity with the stationary and mobile phases
MANNA 23/07/2019 1= Sampling preparation: a key step !
Homogenization liquid-liquid extraction : Folch, Bligh&Dyer…
TLE: total lipid extract Aqueous phase .Water Proteins .Methanol TLE .Chloroform Organic phase Organic phase collected .Water .Alcohol + Internal Standards Quantification TLE Output Supernatant collected
Protein precipitation (plasma)
(Folch J et al, 1957, J. Biol. Chem.) (Bligh EG and Dyer W.J, 1959, Can. J. Biochem. Physiol.) MANNA 23/07/2019 Chromatographic techniques
Liquid Mobile phase Gas
HPLC / UPLC GC
High/ultra performance liquid chomatography Gaz chromatography Capillary column column* SPE Solid Solid phase extraction (polar, ionic, Stationary phase Liquid porous)
plane Plane*
TLC / HPTLC
Thin layer chromatography * cross-linked
Allows the identification and quantification of compounds in a mix
MANNA 23/07/2019 Gas-liquid chromatography (GC): fatty acids
Data Flame ionisation acquisition Injector detector
Oven
Column (stationary phase)
H2 generator (carrier gaz)
MANNA 23/07/2019 FA analysis by gas-liquid chromatography
Separation of FA according to: (= Retention time) - The molecular weight of FA (number of carbons) - The number of double bonds - The cis/trans conformation of double bonds - The position of double bonds
MANNA 23/07/2019 Liquid chromatography on column, SPE
Solid Phase Extraction (SPE) : separation (lipid class), purification or concentration or sampling preparation before analysis
Conditioning Loading Washing Elution Cartridges
Plates 96 wells
Ex separation
MANNA 23/07/2019 High performance liquid chromatography (HPLC)
With detectors currently used in HPLC for lipids: Ultraviolet and visible (UV et DAD), differential refractometry, Light scattering detection (DEDL), Aerosol detectors loaded (Corona)...
Ex plasma Lipid classes
MANNA 23/07/2019 TLC HPTLC Thin layer chromatography High performance - thin layer chromatography
Silica plate
1- Deposit 2 - Migration 3- Revelation
Migration 5. Photography according to the polarity Scanner UV-Visible 4- Visualization
MANNA 23/07/2019 An HPTLC application to separation of lipid classes in plasma of dairy cows and goats fed similar diets
Densitometric analysis
Standard Samples
CE Separation and Identification
TAG FFA Chol PL 0.2 3.3 µL µL Goats Cows
Densitometric analysis via picture
(Delavaud et al. 2019, EJLST, in press) 33 MANNA 23/07/2019 Lipidomic: Techniques of lipid analysis + MS
Sample preparation
• Technique analysis (separation, purification...) SPE LC TLC GC
• Coupled to a mass spectrometer for the identification of unknown compounds
MS analysis and data acquisition
34 MANNA 23/07/2019 Mass spectometry
MANNA 23/07/2019 Mass spectrometer = a detector allowing To conserve the chromatographic resolution a high sensitivity (~ ng) A structural identification A reproducible quantification Schematic representation of all mass detector
Introduction system • Ion generation in a collision (LC, GC, direct = shotgun) cell, synthesis of daughter Under vacuum molecular ions, acceleration of the ions formed Vacuum pump • Mass analyser: separation Ion Mass Detector of these ions followed by source analyzer their detection
Data acquisition Electronic of MS • Data acquistion: Molecular pic reconstruction of the
abundance parent molecule Relative
MANNA 23/07/2019 The mass detector Plateforme MetaToul Separative method
+ +
Separation daughter ions according to their mass on I charge ratio (m / z) m/z m/z m/z (analyzer)
Molecule 1 R T fragmentation Identification of different compounds Reconstruct the parent molecule
MANNA 23/07/2019 Mass spectrometer diversity
Ion sources: Mass analysers: Detectors: -Electron ionisation (EI) - Quadrupole (simple,…) - Electron multiplicator - Chemical ionisation (CI) - Time of flight (TOF) - Electron -MALDI (laser desorption) - Ion trap photomultiplicator or -Electrospray (ESI) - Magnetic sector scintillation detector - APCI (atmospheric - … - Faraday Cylinder… pression ionization) - …
• Choice depends on • Based on the use of • Ion count after signal physicochemical electric and / or amplification properties of the magnetic fields molecules to be studied • Ion separation according to different process
MANNA 23/07/2019 Different combinations for analyses
Coupling GC or HPLC - MS Multi-dimensional analysis MS/MS or MS² or tandem mass spectrometry 3- dimensional informations: several types of associations: 1 = Retention time - 3 quadrupoles QQQ 2 = Intensity or relative abundance 3 = Mass/charge - 1 Q + 1 time of flight (Q-TOF) - 2 TOF (TOF-TOF) - Q and ion trap (IT),…
For structural analysis For high selectivity, high sensitivity, high resolution
XIC : Quantification is performed using the extracted ion chromatograms (XIC) of precursor masses ThermoFisher Scientific From Berdeaux 2014
MANNA 23/07/2019 Two lipidomic approaches can be used: Targeted (type of molecules) or global (profile)
Which one to choose ? TLE
Total lipid extract
Targeted lipidomic approach: Global = a lipidomic profile: With separation… GC ou LC ? ??
With an hypothesis (type of lipids) no apriori
Minor lipids Major lipids
MS low resolution MS high resolution
+ or - separation
Seyer A, Boudah S, Broudin S et al. (2016) Metabolomics 12, 7 MANNA 23/07/2019 Targeted lipidomic by Gas Chromatography methods
Fatty acids Neutral lipids
Sterols
DG
TAG Chol Ester
Oxysterols
5α,6α 7 H H Hβ3-C -EpH3C CH CH O 3 H 3 H CH CH H 3 3
CH3 H CH3 H H3C H3C H H GC-MS Sterols HO OH HO O 7- 6β- Keto H H H3C TriolH3C CH CH 3 H 3 H CH3 CH3 ROS ROS CH3 H CH3 H H3C H3C
H H H H H H3C HO O CH HO 3 2 H 2 OH 1 2 CH3 2 2 OH 1 2 CH3 H 4 ROS 1 0 H3C2 2 H 1 81 1 3 ROS H3C 2 1 5 7 1 1 13 7 2 H CH H 1 3 H 1 9 9 6 H3C CH3 2 1 8 4 6 5 CH3 HO H 3 0 7 CH3 CH3 H 5 H3C 4 6 OH CH3 H H C CYP7A ROS 3 H Cholest 1 érol H H HO OH HO 7 CYP27 CYP46 CYP3A 25 H H H α- OH 4 H3C H3C H3C - O CH CH CH 3 H 3 H 3 H O CH H CH3 3 H OH CH3 H CH3 H CH3 H H3C H3C H3C
H H H H H
HO HO HO
OH 27-OH 24(S)- 4 OH β- O H
MANNA 23/07/2019 Targeted lipidomic by Liquid Chromatography methods
Bile acids Sphingolipids Phospholipids Major Major
Ceramide,Sphingomyeline, LC-MS GluCer, LactCer… PE, PC, PI, PS Minor Minor
LPA, LPC, PIPs PUFA metabolites So, Sa, SoP Eicosanoids Isoprostane Thromboxane A1 OH HO Prostaglandin E1 (PGE2) OH CO2H HO 5 COOH O O CO2H 5 COOH 20 O 20 HO 5-F -IsoP OH 2t HO HO OH 5-F3t-IsoP
MANNA 23/07/2019 Global: Direct analysis by shot gun
Definition: direct injection of a lipid extract sample
I
Direct analysis by MS
m/z Sample
Schematic outline of a high-throughput shotgun lipidomic platform:
Quick Suitable for big series
Major lipids Heavy data processing
Ståhlman M, Ejsing CS, Tarasov K et al. (2009) High-throughput shotgun lipidomics by quadrupole time-of-flight mass spectrometry. Journal of Chromatography B 877, 2664-2672. MANNA 23/07/2019 Global: Analysis with separation by LC Plateforme MetaToul Chromatographic separation
Mode pos/Neg
Columns: HILIC or Silica pure-> by class
Database needed
Heavy data processing
No minority species detected
Baker, JLR, 2014 (ex HILIC-UPLC-MS) Seyer et al. 2016 (LC-HRMS)
MANNA 23/07/2019 Quantification in lipidomic
Absolute quantification is:
1 internal standard + 1 primary standard OH OH
COOH COOH OH D OH D D Complex Lipids: OH D D OH 5 LXA4 LXA4 d Numerous molecular = a calibration curve 10 Area species y = ax +b signal x ISTD x x x 5 x x x Lack of all standards (internal calibration with 0 0 200 400 600 limited set STD) Quantity injected Response of the MS detector/molecular species can vary
Relative Quantification
MANNA 23/07/2019 A schematic Workflow (from sample preparation to analysis of the data)
Schematic lipidomics analysis workflow
Sampling, storage, lipid Analysis: GC-MS, LC-MS, Data processing extraction shotgun..
Lipid database
• Lipid extraction* using • Ex. LC and MS defined organic solvant parameters and raw • Analysis of • Structural characterization and *Folch (1957), Bligh and Dyer (1959), data acquisition (peak fragmentation identification of lipid species MTBE, BUME methods.... detection, integration) (Spectral data analysis) • Bioinformatics • Derivation... • MS data acquisition
=> Biological interpretation and possible applications (Biomedical, animal science..) Identification of molecular mechanisms...
See Yang K, Han X (2016) Lipidomics: Techniques, Applications, and Outcomes Related to Biomedical Sciences. Trends in Biochemical Sciences 41, 954-969. MANNA 23/07/2019 Some available resources: Public or commercial
MANNA 23/07/2019 http://www.lipidmaps.org/resources/tutorials/videos. php
48 MANNA 23/07/2019 Conclusions on lipidomics
Lipidomics aim to analyze a set of molecules and their integration represent a powerful framework for studying specific phenotypic traits and mechanisms… Like other "omics" technologies, none of them alone can satisfactorily answer a biological question Must be combined with different omic approaches and levels of study (animal, tissue, cell ...) to allow an integrative analysis of metabolism A "case-by-case" deepening is essential to arrive at integrated biological concepts
MANNA 23/07/2019 _03 Lipidomics: example of applications
.050 Some publications on lipidomics (lots in human): Mainly on methods, pathology (metabolism, disease...) => biomarker research
Benedusi V (2018) Lipidomics. Materials and Methods, vol. 8., DOI: 10.13070/mm.en.8.2665 Griffiths WJ, Wang Y (2009) Mass spectrometry: from proteomics to metabolomics and lipidomics. Chem Soc Rev 38, 1882-1896.
Lam SM, Shui GH (2013) Lipidomics as a Principal Tool for Advancing Biomedical Research. J Genet Genomics 40, 375-390. Seyer A, Boudah S, Broudin S et al. (2016) Annotation of the human cerebrospinal fluid lipidome using high resolution mass spectrometry and a dedicated data processing workflow. Metabolomics 12, 7.
Postle AD (2009) Phospholipid lipidomics in health and disease. Eur J Lipid Sci Tech 111, 2-13. Quehenberger O, Armando AM, Brown AH et al. (2010) Lipidomics reveals a remarkable diversity of lipids in human plasma. J Lipid Res 51, 3299-3305.
Schwaiger M, Schoeny H, El Abiead Y et al. (2019) Merging metabolomics and lipidomics into one analytical run. Analyst 144, 220-229.
Messias MCF, Mecatti GC, Angolini CFF et al. (2018) Plasma lipidomic signature of rectal adenocarcinoma reveals Potential Biomarkers. Front Oncol 7, 9.
Wood PL, Cebak JE (2018) Lipidomics biomarker studies: Errors, limitations, and the future. Biochemical and biophysical research communications 504, 569-575. Key words: Signature, characterization, biomarkers MANNA 23/07/2019 A non-exhaustive list of selected recent Applications of Lipidomics for Biological and Biomedical Researches (reviewed by Yang et al., 2016)
Metabolic Neurological Cancer Eye disease syndrome disorders • Cardiovascular • Alzheimer's • Breast, prostate; • Vision research disease (CVD) disease lung • Tear analysis • Inflammatory • Huntington • ovary; inocular surface diseases (e.g., disease • esophagus; diseases atherosclerosis • Multiple sclerosis • kidney; skin • Meibomian gland • HDL-based secretion (MGS) therapies in and tear film atherosclerotic inocular surface vascular disease protection • Diabetes and • Inflammation and obesity neuroprotection Report: • techniques used, source Multiomics of lipids (biological Drug discovery/ Nutrition and personalized sample origin) screen medicine • Findings and • Dietary intervention • Target discovery; • Type 1 diabetes (T1D) • Health benefits of • candidate drugs; • asthma; implications described dietary ω-3 PUFA • preclinical/clinical • chronic pain ; (references to the • testing; toxicity; • global lipid metabolism reviews) • Biomarkers efficacy; ... • systems and dynamical medicine; diet effects
Yang K, Han X (2016) Lipidomics: Techniques, Applications, and Outcomes Related to Biomedical Sciences. Trends in Biochemical Sciences 41, 954-969. Lam SM, Shui GH (2013) Lipidomics as a Principal Tool for Advancing Biomedical Research. J Genet Genomics 40, 375-390. MANNA 23/07/2019 Studies using lipidomics in animal studies: Applications
Characterization of products (milk, meat,...), tissue, biological fluid Authentification of products
Lipidome response to: Nutrition Pathology (steatosis, insulin resistance, inflammation....) Disease
MANNA 23/07/2019 Few recent publications on lipidomics for characterisation of the animal milk lipidome Li Q, Zhao Y, Zhu D et al. (2017) Lipidomics profiling of goat milk, soymilk and bovine milk by UPLC-Q-Exactive Orbitrap Mass Spectrometry. Food Chemistry 224, 302-309. Castro-Gomez P, Montero O, Fontecha J (2017) In-Depth Lipidomic Analysis of Molecular Species of Triacylglycerides, Diacylglycerides, Glycerophospholipids, and Sphingolipids of Buttermilk by GC-MS/FID, HPLC-ELSD, and UPLC-QToF-MS. Int J Mol Sci 18.
Liu Z, Rochfort S, Cocks B (2018) Milk lipidomics: What we know and what we don't. Prog Lipid Res 71, 70-85. Ding F, Cousillas GT, Weber WJ et al. (2016) Effect of milk yield genotype on lipidomic profiles of multiparous Holstein cows during the first 9 wk of lactation. Journal of animal science 94, 55-55. George AD, Gay MCL, Trengove RD et al. (2018) Human Milk Lipidomics: Current Techniques and Methodologies. Nutrients 10 Yassin AM, AbdelTeng Hamidet al. 2019:MI, Farid A method OA et al. combined(2016) Dromedary the solid phasemilk exosomes extractionas mammary transcriptome(SPE),nano -highvehicle-performance: Their isolation, liquid vesicular chromatographyand phospholipidomic (HPLC) and characterizations. Journal of Advanced Research 7, 749-756. multi-dimension mass spectrometry (MDMS) developed => up to Teng F, Reis MG,105, Yang 64, 14L et and al. (2019)30 species In-depth of TAGs,lipidomic DAGs,analysis MAGs, ofand tri -FFAs, di- , wereand mono - acylglycerolsdeterminedreleased from withmilk concentrationsfat after in vitro from digestion. 0.01 to Food 191.0 Chemistry mg/g fat,297, 12. La Nasa J, DeganoduringI, Brandolinithe in vitroL digestionet al. (2018) of Acow novel andHPLC sheep-ESI milk-Q-ToF approach for the determination of fatty acids and acylglycerols in food samples. Anal Chim Acta 1013, 98-109. (milk) MANNA 23/07/2019 List of publications on lipidomics approaches in animal studies: Product Authentification
Cossignani L, Pollini L, Blasi F (2019) Invited review: Authentication of milk by direct and indirect analysis of triacylglycerol molecular species. Journal of Dairy Science 102, 5871-5882.
Hrbek V, Ovesna J, Demnerova K et al. (2015) Utilization of Supercritical Fluid Chromatography for Lipidomic Profilation of Soya Bean and Cow Milk: Authenticity and Detection of Adulteration. Chem Listy 109, 518-526.
MANNA 23/07/2019 List of few publications of lipidomics approaches in animal studies: Veterinary studies (pathology, disease)
Equine
Elzinga S, Wood P, Adams AA (2016) Plasma Lipidomic and Inflammatory Cytokine Profiles of Horses With Equine Metabolic Syndrome. Journal of Equine Veterinary Science 40, 49-55. de Grauw JC, van de Lest CHA, van Weeren PR (2011) A targeted lipidomics approach to the study of eicosanoid release in synovial joints. Arthritis Res Ther 13, R123.
de Grauw JC, van de Lest CHA, van Weeren PR (2011) A targeted lipidomics approach to the study of eicosanoid release in synovial joints. Arthritis Res Ther 13, R123.
Feline Gray-Edwards HL, Jiang X, Randle AN et al. (2017) Lipidomic Evaluation of Feline Neurologic Disease after AAV Gene Therapy. Mol Ther Methods Clin Dev 6, 135-142.
MANNA 23/07/2019 _04 Lipidomics: An example of application in dairy animals
.057 Milk molecular species of triacylglycerols and polar lipids characterized by lipidomic approach in cows and goats fed diets supplemented with various lipids sources H. Fougère, C. Delavaud, J. Bertrand-Michel, L. Bernard UMR1213 Herbivores, INRA Vetagro Sup, F-63122 St-Genès-Champanelle, France
NOM DE L’AUTEUR JOUR / MOIS / ANNEE => Lipid fraction is a major determinant of milk nutritional quality and efficiency of production that can be modulated by nutritional factors such as lipid supplementation
Project with objective : determine the key drivers of milk fat secretion
Approach: Use of a model of comparative biology to study the mechanisms of lipid metabolism regulation among dairy cows and goats • A comparative study between dairy cows and goats fed similar diets supplemented with various lipids • Lipidomic targeted approach to characterize milk composition molecular species of triacylglycerols (TAG) and polar lipids. • In complement of GC (for FA) + HPLC (for CLA isomers)
59 MANNA 23/07/2019 Lipid supplements chosen: 2 types diets that induce a milk fat depression MFD in dairy cows: rich in concentrate + PUFA Vegetable oil + starch Marine product 1 diet to increase the milk fat content in dairy cows: rich SFA Vegetable oil
Experimental design: 2 Replicated 4 x 4 Latin Square 4 periods of 28-d, 4 diets based on 45% Forage + 55% Concentrate: N = 12 N = 12
. Control (CTL): not supplemented in lipid
. Corn oil plus starch (COS): + Corn Oil (5% DMI) rich in 18:2n-6 + Starch
. Marine algae powder (MAP): + Schyzochitrium sp (1.5% DMI) rich in 22:6 n-3 (DHA)
. Hydrogenated palm oil (HPO): + Hydrogenated Palm Oil (3% DMI) rich in 16:0 et 18:0
60 MANNA 23/07/2019 Results of dairy performance: milk fat content
Fat content COS MAP HPO
-45% -22% +13%
= -15% =
. Diet effects in accordance with our hypothesis . Strong differences among species with diets that ↑ or ↓ milk fat content
An original experimental model to study lipid metabolism
Fougère et al., 2018. Journal of Dairy Science 101 (9):8429–8445.
61 MANNA 23/07/2019 Why Lipidomic?
• Characterize the milk fat composition in molecular species of TAG and polar lipids • to decipher the mechanims of the differences of lipid metabolism regulation among these 2 species • Knowledge on quality of milk lipid fraction (TAG and polar lipids: health benefits and technological properties) according to diet and species
62 MANNA 23/07/2019 MS Sample Lipid Biological Analysis/data collection extraction interpretation Workflow acquisition
• 2 consecutive • Bligh and Dyer, • TAG LC-HR/MS • Data processing milking on d24 1959 • Polar lipids LC-ESI- (Anova, PCA, • Pooled per period, MS/MS statistics,...) diet, species = 16 • In complement samples/species with data GC and HPLC
Reminder: Milk fat = TAG (98 %) + polar lipids (≈ 1 %)
TAG Polar lipids Fatty acids composition Phosphatidylcholine FA1 Phosphatidylethanolamine . GC Phosphatidylserine FA2 . HPLC: + CLA isomers Phosphatidylinositol FA . Lipidomic approach 3 Sphyngomyelin Ceramides TAG (X:Y) (Not reported)
Number of total C Number of total unsaturation 63 MANNA 23/07/2019 Results of milk TAG molecular species 48 TAG molecular species : from (18:0) to TAG (64:3) identified in milk of cows and goats fed various lipid supplement Species specificities of response to diets for 19 TAG among the 48
On going analysis: first results on milk TAG (PCA)
Species effect on milk TAG Dietary effect on milk TAG
Goat Cow
64 MANNA 23/07/2019 TAG molecular species and selected FA, relation with fat content (PCA)
Fatty acids, TAG PC2 (25% PC2 (25% )
Fat content
PC1 (32%) Fat content negatively related to TAG molecular species (TAG 50:3, TAG 54:2, 54:3, 56:2...)
65 MANNA 23/07/2019 TAG molecular species and FA negatively related with fat content
(PCA) PC2 (25% PC2 (25% )
PC1 (32%)
These antilipogenic fatty acids linked to these TAG molecular species?
66 MANNA 23/07/2019 Contribution of lipidomic approach to our project
Means for analysing milk lipids
GC (FA HPLC analysis) (CLA) )
Targeted lipidomics (TAG, PL)
Hypothesis on molecular FA linked to TAG species and related to milk fat secretion Characterization of milk TAG and polar lipids molecular species according to species and diet => authentification? Biomarker? (species, diet...)
67 MANNA 23/07/2019 Lipidomics: key points
• Lipidomics allow the characterization of metabolic pathways of lipid classes, subclasses and individual species to help understand lipid metabolism • Applications of lipidomics for studying lipid metabolism increase our understanding of the molecular mechanisms involved
• A truly comprehensive lipidomic analysis (particularly for those lipids in very low abundance) in an automated, high-throughput manner remains demanding
Perspectives:
Increased knowledge of lipid metabolism pathways and networks, and bioinformatics tools are essential for studying lipid metabolism using lipidomics Integration of lipidomics data with other -omics strategies to maximize the power of this method Develop tools that can integrate lipidomics data with gene, transcript and enzymatic data to enable metabolic pathway reconstruction and flux analyses, whereby stable isotopes are used to study metabolic pathways
68 MANNA 23/07/2019 Different levels of approach => An integrative analysis of metabolism
Tissue, biological fluid
Omics -Lipidomic -Metabolomic -Transcriptomic Whole animal - Proteomic -.. Cell Molecules (Lipids, genes, prot...)
69 MANNA 23/07/2019 Thank you for your attention ! .070