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NOM DE L’AUTEUR JOUR / MOIS / ANNEE
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
2
What ?
Where ?
Why ?
How?
Lipids
Definition and structure: molecular species
Location, biological functions
Interest in lipid determination
Techniques for lipid analysis => Lipidomics
Case study
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An application in dairy animals
Will address the following...
Presentation
.03
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
Definition and structure of lipids:
molecular species
_01
.04
.
Fahy, E. et al., 2005, Eur J Lipid Sci Tech
Lipid: Definition
• Lipids have been globally defined as biological substances that are generally
hydrophobic in nature and in many cases soluble in organic solvents
=> These chemical properties cover a broad range of molecules, such as
fatty acids, phospholipids, sterols, sphingolipids, terpenes, and others
No agreement in the lipid community:
• 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.)
What?
MANNA 23/07/2019• Christie et al., 2003
Lipids
• In various living organisms
• 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)
Where?
Why?
=> Key role in various diseases (metabolic, cancer, cardiovascular, inflammation...) and are impacted by nutrition
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• In all animal tissues and biological fluids
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Lipids: molecular species in 8 categories
Glycerol
FA
cholesterol
Simple
lipids
Consistent structure representation across categories
Fatty Acyls (FA)
Sterol Lipids (ST)
Prenol Lipids (PR)
Glycerolipids (GL)
OH
O
O
O
HO
OH
O
O
H
H
H H
H
O
Phosphate
Amide linkage
Complex
lipids
Glycerophospholipids (GP)
Sphingolipids (SP)
O
O
HO
O ON+
P
-O
O
O
OH
NH
OHH
O
H
Saccharolipids (SL), Polyketides (PK)
(Lipid Maps Lipid Chemistry Tutorial)
(Triacylglycerides;TAG)
(Phospholipids; PL)
An extensive range of quantities
TAG
CEPC
Chol(FA) Fatty acids
(SP) Sphingolipid LPC
Cholesterol ester (ST)
(PL) Phosphatidylcholine
(from Christie et al, JLR, 1985) Example of Lipid composition human plasma
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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.
Triacylglyceride (GL)
=> Modulated according to the species(Kaabia et al., 2018)
Sphingolipid (≈2%)
(Hammad SM et al. J. Lipid Res. 2010;51:3074-3087)
An extensive range of quantities
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Sphingolipids
Sphingomyelin
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Lipidomics reveals a remarkable diversity of lipids in human plasma:
Quehenberger et al. (2010)
An extensive range of molecules
(TAG,...) (PL)
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Structure of the fatty acids:
• Saturated
CH3
Palmitic acid – 16:0
12
3COOH
16
1
• Branched chain fatty acids, hydroxyded fatty acids acids...
Oleic acid – 18:1 cis9 (ω9)
CH3COOH
9
18
6
Linolenic acid – 18:3 cis9 cis12 cis15 (ω3)
CH39
COOH12 1815
3
• Ex of mono and polyunsaturatedof C18
Linoleic acid – 18:2 cis9 cis12 (ω6)
CH3
9COOH
12
186
= hydrocarbonated aliphatic chain; with a carboxylicfunction; 2 to 24 atoms of C; with or without double bond
Conjugated LinoleicAcid (CLA)
Rumenic acid – 18:2 cis9 trans11, c9t11-CLA9
CH3COOH18
11
CLA:
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Structure of the fatty acids: Usual nomenclatures
Example of Linoleic acid
18:2 n-6 (or w6) or 18:2 Δ9,12 (cis9,cis12-18:2)
Position of the first double bond from the methyl end
Position of double bonds from the carboxyl end
Number of double bonds
Number of carbons
n or
Δ CH318
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Trans configuration :(bacterial or technological origin)
The two hydrogen atoms are bound to opposite sides of the double bond.
The FA chain is not curved
Elaidic acid (18:1 trans9, n-9)
Oleic acid (18:1 cis9 or n-9)
Adjacent hydrogen atoms are on the same side of the double bond.
The FA chain is curved
Cis configuration :
Melting point: 13 °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)…
Melting point: 44°C
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28 possible configurations (isomers)
numerous biological properties(cancers, obesity, antilipogenic…)
Linoleic acidc-9, c-12 isomer
CLA c-9, t-11 isomer(rumenic acid)
Conjugated linoleic acid (CLA)(in ruminant products)
12
6
11
Rumenic acid – 18:2 cis9 trans11, c9t11-CLA9
CH3COOH18
11
Linoleic acid – 18:2 cis9 cis12 (ω6)
CH3
9COOH
12
186
9
Lipid MAPS Website: a huge source of information
on lipidshttps://www.lipidmaps.org/
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http://www.lipidmaps.org/resources/databases/index.php?tab=lmsd
Search basedon
structure....
LIPID MAPS Structure Database (LMSD)
http://www.lipidmaps.org/data/structure/
Online functions:
Browse
Text search
Substructure search
Download structures
Other Websites...
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 bibliographydevoted to the main presented topics.
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=> provide useful online resources for an overview of these molecules and their
structures and methods for analysis
Lipidomics
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Genomics
Transcriptomics
Proteomics
Metabolomics
Bioinformatics
Lipidomics
Omics Technologies: a field of study in Biology
Is the part of metabolomics dedicated to lipids: the full molecularcharacterization of the lipid components in a defined system, as well as consideration of their function when available
(Liu et al, 2018 Progress in Lipid Research 71,70-85)
(Lagarde et al, 2003 BBA)
This term used in the titles of scientificarticles: 1st in 2002, ≈ 300 the last 2 years
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
• Allow a comprehensive analysis of lipid molecules
Lipidomics: Definition and key points
(Fahy et al. 2005, EJLST)
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A discipline at the interface of lipid biology technology, biomedical science and animal science:
(Lam and Shui, 2013 J Genet Genomics)
≈ 300
• Lipidomics is the study of lipidomes (= a large variety of molecules with variouspattern of concentration)
2002 2018
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Lipidomics: How?
Uses the techniques of analytical chemistry to study the entirelipid content of a cell, a tissue, a biological fluid...
Associated with mass spectrometry (MS): liquid-chromatography MS, shotgun lipidomics...
=> An emerging discipline that evolved thanks to the increase in the analytical power of mass spectrometry (MS) and new developments in MS
=> By techniques of lipid analysis + MS
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Techniques of lipid Analysis
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
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How?
1= Sampling preparation: a key step !
+ InternalStandards
QuantificationOutput
Homogenization
WaterAlcohol
TLE
Supernatantcollected
Protein precipitation (plasma)
(Folch J et al, 1957, J. Biol. Chem.) (Bligh EG and Dyer W.J, 1959, Can. J. Biochem. Physiol.)
TLE
Organicphase
collected
Aqueousphase
Proteins
Organicphase
WaterMethanolChloroform
liquid-liquid extraction : Folch, Bligh&Dyer…
TLE: total lipid extract
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Stationary phase Liquid
Solid
(polar, ionic, porous)
Liquid GasMobile phase
Chromatographic techniques
Allows the identification and quantification of compounds in a mix
HPLC / UPLC GC
columnSPE
Capillarycolumn*
High/ultra performance liquid chomatography
Solid phase extraction
Gaz chromatography
TLC / HPTLC
planePlane*
* cross-linkedThin layer chromatography
Gas-liquid chromatography (GC): fatty acids
Data acquisition
Flame ionisationdetector
H2 generator(carrier gaz)
Injector
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Oven
Column(stationary
phase)
Separation of FA according to:
- 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
FA analysis by gas-liquid chromatography
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(= Retention time)
Conditioning Loading Washing Elution
Solid Phase Extraction (SPE) : separation (lipid class), purification or concentration or sampling preparation before analysis
Cartridges
Plates 96 wells
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Liquid chromatography on column, SPE
Ex separation
High performance liquid chromatography (HPLC)
With detectors currently used in HPLC for lipids: Ultraviolet and visible (UV et DAD), differential refractometry, Light scatteringdetection (DEDL), Aerosol detectors loaded(Corona)...
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Ex plasma Lipid classes
Silicaplate
Thin layer chromatography
Migration according to the polarity
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TLC HPTLCHigh performance -
thin layer chromatography
1- Deposit2 - Migration
3- Revelation
Scanner UV-Visible
5. Photography
4- Visualization
33
Densitometric analysis
Separation and Identification
Densitometric analysisvia picture
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An HPTLC application to separation of lipid classes in plasma of dairy
cows and goats fed similar diets
Cows
CE
TAG
FFA
Chol
PL
Standard Samples
Goats
0.2µL
3.3µL
34
Lipidomic: Techniques of lipid analysis + MS
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• Coupled to a mass spectrometer for the identification of unknown compounds
• Technique analysis (separation, purification...)
GC SPELC
MS analysis and data acquisition
TLC
Sample preparation
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Mass spectometry
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Mass spectrometer = a detector allowing
To conserve the chromatographic resolution a high sensitivity (~ ng) A structural identification A reproducible quantification
Ion source
Vacuum pump
Mass analyzer
Detector
Electronic of MS
Under vacuum
Introduction system (LC, GC, direct = shotgun)
Data acquisitionMolecular
pic
Rel
ativ
e ab
un
dan
ce
Schematic representation of all mass detector
• Data acquistion: reconstruction of the parent molecule
• Mass analyser: separation of these ions followed by their detection
• Ion generation in a collision cell, synthesis of daughtermolecular ions, acceleration of the ions formed
+ +
Plateforme MetaToulThe mass detector
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Separative method
Identification of
different compounds
Reconstruct the parent molecule
I
R
T
m/z m/z m/z
Molecule 1 fragmentation
Separation daughter ions according to their mass on charge ratio (m / z) (analyzer)
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Mass spectrometer diversity
Ion sources:-Electron ionisation (EI)- Chemical ionisation (CI)-MALDI (laser desorption)-Electrospray (ESI)- APCI (atmospheric
pression ionization)- …
Mass analysers:- Quadrupole (simple,…)- Time of flight (TOF)- Ion trap- Magnetic sector- …
Detectors:- Electron multiplicator- Electron
photomultiplicator or scintillation detector
- Faraday Cylinder…
• Choice depends on physicochemical properties of the molecules to be studied
• Based on the use of electric and / or magnetic fields
• Ion separation according to different process
• Ion count after signal amplification
Coupling GC or HPLC - MS
3- dimensional informations:
1 = Retention time2 = Intensity or relative abundance3 = Mass/charge
From Berdeaux 2014
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Different combinations for analyses
Multi-dimensional analysis
MS/MS or MS² or tandem mass spectrometryseveral types of associations:
- 3 quadrupoles QQQ
- 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 massesThermoFisher Scientific
TLEWhich one to choose ?
Total lipidextract
?? Global = a lipidomic profile:
no apriori
Major lipids
MS high resolution
+ or - separation
Targeted lipidomic approach:
With separation… GC ou LC ?
With an hypothesis (type of lipids)
Minor lipids
MS low resolution
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Seyer A, Boudah S, Broudin S et al. (2016) Metabolomics 12, 7
Two lipidomic approaches can be used:
Targeted (type of molecules) or global (profile)
Targeted lipidomic by Gas Chromatography methods
Neutral lipids
ROS
ROSROS
ROS
CYP7A1
CYP27 CYP4625-OH
H
CH3
HCH3
HO
H
H
H3C
H
CH3
H3COH
27-OH
H
CH3
HCH3
HO
H
H
H3C
H
OH
H3C
24(S)-OH
CH3
H3C
H
CH3
HCH3
HO
H
H
H3C
H
OH
7-Keto H
CH3
HCH3
HO
H
H
H3C
H
O
CH3
H3C
5α,6α-Ep
CH3
H3C
H
CH3
HCH3
HO
H
H
H3C
O
7α-OH
CH3
H3C
H
CH3
HCH3
HO
H
H
H3C
OH
7β-OH
CH3
H3C
H
CH3
HCH3
HO
H
H
H3C
OH
4β-OH
CH3
H3C
H
CH3
HCH3
HO
H
H
H3C
OH
6β-Triol
H
CH3
HCH3
HO
H
H
H3C
H
CH3
H3C
OH
OH
CH3
H3C
H
CH3
HCH3
HO
H
H
H3C
Cholestérol
123
45
6
78
910
11
12
1314
15
16
171
9
18
20
21
22
23
242
527
26
CYP3A4
ROS
Oxysterols
Fatty acids
DG
TAGChol Ester
Sterols
Sterols
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GC-MS
Targeted lipidomic by Liquid Chromatography methods
PhospholipidsSphingolipids
Major
Minor Minor
Major
PUFA metabolites
Thromboxane A1
O
OH
COOHO
Prostaglandin E1 (PGE2)
OHOH
COOH
O
Eicosanoids Isoprostane
HO
HO
OH
CO2H5
20
5-F3t-IsoP
HO
HOOH
5
20
5-F2t-IsoP
CO2H
PE, PC, PI, PS
LPA, LPC, PIPs
Ceramide,Sphingomyeline, GluCer, LactCer…
So, Sa, SoP
Bile acids
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LC-MS
Definition: direct injection of a lipid extract sample
Direct analysis by MS
I
m/zSample
Global: Direct analysis by shot gun
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Schematic outline of a high-throughput shotgun lipidomic platform:
Ståhlman M, Ejsing CS, Tarasov K et al. (2009) High-throughput shotgun lipidomicsby quadrupole time-of-flight mass spectrometry. Journal of Chromatography B877, 2664-2672.
QuickSuitable for big series
Major lipidsHeavy data processing
Baker, JLR, 2014 (ex HILIC-UPLC-MS)
Chromatographic separation
Mode pos/Neg
Columns: HILIC or Silicapure-> by class
Database needed
Heavy data processing
No minority speciesdetected
Plateforme MetaToul
Global: Analysis with separation by LC
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Seyer et al. 2016 (LC-HRMS)
Quantification in lipidomic
Absolute quantification is:
OH
OHCOOH
OH
LXA4
OH
OHCOOH
OH D D
D
DD
LXA4 d5
0
5
10
0 200 400 600Quantity injected
Area signal
y = ax +b
xx
xx
xx
xISTD
1 internal standard + 1 primary standard
= a calibration curve
Complex Lipids:
Numerous molecularspecies
Lack of all standards (internal calibration withlimited set STD)
Response of the MS detector/molecular speciescan vary
Relative Quantification
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Schematic lipidomics analysis workflow
Sampling, storage, lipidextraction
Analysis: GC-MS, LC-MS, shotgun..
Data processing
• Ex. LC and MS definedparameters and rawdata acquisition (peakdetection, integration)
• MS data acquisition
=> Biological interpretation and possible applications (Biomedical, animal science..)
Identification of molecular mechanisms...
• Lipid extraction* usingorganic solvant
*Folch (1957), Bligh and Dyer (1959),MTBE, BUME methods....
• Derivation...
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• Structural characterization and identification of lipid species
• Bioinformatics
Lipid database
• Analysis of fragmentation (Spectral data analysis)
A schematic Workflow (from sample preparation to analysis of the data)
See Yang K, Han X (2016) Lipidomics: Techniques, Applications, and Outcomes Related to Biomedical Sciences. Trends in Biochemical Sciences 41, 954-969.
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Some available resources: Public or commercial
48
http://www.lipidmaps.org/resources/tutorials/videos.
php
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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
Conclusions on lipidomics
Lipidomics: example of applications
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Some publications on lipidomics (lots in human): Mainly on methods, pathology
(metabolism, disease...) => biomarker research
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.
Griffiths WJ, Wang Y (2009) Mass spectrometry: from proteomics to metabolomics and lipidomics. ChemSoc Rev 38, 1882-1896.
Benedusi V (2018) Lipidomics. Materials and Methods, vol. 8., DOI: 10.13070/mm.en.8.2665
Messias MCF, Mecatti GC, Angolini CFF et al. (2018) Plasma lipidomic signature of rectal adenocarcinomareveals 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
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A non-exhaustive list of selected recent Applications of Lipidomics for Biological and Biomedical Researches (reviewed by Yang et al., 2016)
Nutrition
• Dietary intervention
• Health benefits of dietary ω-3 PUFA
Drug discovery/
screen
• Target discovery;
• candidate drugs;
• preclinical/clinical
• testing; toxicity;
• Biomarkers efficacy; ...
Multiomics
and personalized
medicine
• Type 1 diabetes (T1D)
• asthma;
• chronic pain ;
• global lipid metabolism
• systems and dynamicalmedicine; diet effects
Yang K, Han X (2016) Lipidomics: Techniques, Applications, and Outcomes
Related to Biomedical Sciences. Trends in Biochemical Sciences 41, 954-969.
Metabolicsyndrome
• Cardiovasculardisease (CVD)
• Inflammatorydiseases (e.g., atherosclerosis
• HDL-basedtherapies in atheroscleroticvascular disease
• Diabetes and obesity
Neurologicaldisorders
• Alzheimer'sdisease
• Huntington disease
• Multiple sclerosis
Cancer
• Breast, prostate; lung
• ovary;
• esophagus;
• kidney; skin
Eye disease
• Vision research
• Tear analysisinocular surface diseases
• Meibomian gland secretion (MGS) and tear film inocular surface protection
• Inflammation and neuroprotection
Lam SM, Shui GH (2013) Lipidomics as a Principal Tool for Advancing
Biomedical Research. J Genet Genomics 40, 375-390.
Report: • techniques used, source
of lipids (biologicalsample origin)
• Findings and implications described(references to the reviews)
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Studies using lipidomics in animal studies:
Applications
Characterization of products (milk, meat,...), tissue, biological fluid
Authentification of products
Lipidome response to: NutritionPathology (steatosis, insulin resistance,
inflammation....)Disease
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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 milkby UPLC-Q-Exactive Orbitrap Mass Spectrometry. Food Chemistry 224, 302-309.
Liu Z, Rochfort S, Cocks B (2018) Milk lipidomics: What we know and what we don't. Prog Lipid Res 71, 70-85.
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.
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, Abdel Hamid MI, Farid OA et al. (2016) Dromedary milk exosomes as mammary
transcriptome nano-vehicle: Their isolation, vesicular and phospholipidomic characterizations. Journal of Advanced Research 7, 749-756.
Teng F, Reis MG, Yang L et al. (2019) In-depth lipidomic analysis of tri-, di-, and mono-
acylglycerols released from milk fat after in vitro digestion. Food Chemistry 297, 12.
La Nasa J, Degano I, Brandolini L et al. (2018) A novel HPLC-ESI-Q-ToF approach for the
determination of fatty acids and acylglycerols in food samples. Anal Chim Acta 1013, 98-109.
(milk)
Teng et al. 2019: A method combined the solid phase extraction(SPE), high-performance liquid chromatography (HPLC) and multi-dimension mass spectrometry (MDMS) developed => up to 105, 64, 14 and 30 species of TAGs, DAGs, MAGs, and FFAs were determined with concentrations from 0.01 to 191.0 mg/g fat, during the in vitro digestion of cow and sheep milk
List of publications on lipidomics approaches in animal studies:
Product Authentification
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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.
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.
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Elzinga S, Wood P, Adams AA (2016) Plasma Lipidomic and Inflammatory Cytokine
Profiles of Horses With Equine Metabolic Syndrome. Journal of Equine Veterinary Science40, 49-55.
List of few publications of lipidomics approaches in animal studies:
Veterinary studies (pathology, disease)
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.
Equine
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.
Gray-Edwards HL, Jiang X, Randle AN et al. (2017) Lipidomic Evaluation of FelineNeurologic Disease after AAV Gene Therapy. Mol Ther Methods Clin Dev 6, 135-142.
Feline
Lipidomics: An example of application
in dairy animals
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NOM DE L’AUTEUR JOUR / MOIS / ANNEE
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
59
• 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)
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Project with objective : determine the key drivers of milk fat secretion
=> 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
Approach: Use of a model of comparative biology to study the mechanisms of lipid metabolism regulation among dairy cows and goats
60MANNA 23/07/2019
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
Lipid supplements chosen:
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
N = 12
2 Replicated 4 x 4 Latin Square
4 periods of 28-d, 4 diets based on 45% Forage + 55% Concentrate:
N = 12
Experimental design:
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Fat content COS MAP HPO
-45% -22% +13%
= -15% =
Results of dairy performance: milk fat content
An original experimental model to study lipid metabolism
Fougère et al., 2018. Journal of Dairy Science 101 (9):8429–8445.
Diet effects in accordance with our hypothesis Strong differences among species with diets that ↑ or ↓ milk fat content
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Why Lipidomic?
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• 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
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WorkflowSample
collection
• 2 consecutivemilking on d24
• Pooled per period, diet, species = 16 samples/species
Lipidextraction
• Bligh and Dyer, 1959
MS Analysis/data
acquisition
• TAG LC-HR/MS
• Polar lipids LC-ESI-MS/MS
Biologicalinterpretation
• Data processing(Anova, PCA, statistics,...)
• In complementwith data GC and HPLC
Fatty acids composition GC HPLC: + CLA isomers
TAG
FA1
FA2
FA3
Reminder: Milk fat = TAG (98 %) + polar lipids (≈ 1 %)
PhosphatidylcholinePhosphatidylethanolaminePhosphatidylserinePhosphatidylinositolSphyngomyelinCeramides
Polar lipids
(Not reported)TAG (X:Y) Lipidomic approach
Number of total C Number of total unsaturation
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Dietary effect on milk TAG
On going analysis: first results on milk TAG (PCA)
Species effect on milk TAG
GoatCow
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
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TAG molecular species and selected FA, relation with fat content
(PCA)
Fat content negatively related to TAG molecular species (TAG 50:3, TAG 54:2, 54:3, 56:2...)
Fat content
Fatty acids, TAG
PC1 (32%)
PC
2 (
25%
)
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These antilipogenic fatty acids linked to these TAG molecular species?
PC1 (32%)
PC
2 (
25%
)
TAG molecular species and FA negatively related with fat content
(PCA)
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Contribution of lipidomic approach to our project
Means for analysing milk lipids
GC (FA
analysis))
HPLC
(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...)
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Lipidomics: key points
• Lipidomics allow the characterization of metabolic pathways of lipid classes, subclasses and individual species to help understand lipid metabolism
• A truly comprehensive lipidomic analysis (particularly for those lipids in very low abundance) in an automated, high-throughput manner remains demanding
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
Perspectives:
• Applications of lipidomics for studying lipid metabolism increase our understanding of the molecular mechanisms involved
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Different levels of approach
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Whole animal
Cell
Molecules(Lipids, genes,
prot...)
Tissue, biological fluid
Omics
-Lipidomic
-Metabolomic
-Transcriptomic
- Proteomic
-..
=> An integrative analysis of metabolism
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Thank you for your attention !
