Lipidomics ERN 4221 9107

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Lipidomics ERN 42219/1-07

• Christian A. Drevon
• Department of Nutrition
• Institute of Basic Medical Sciences
• University of Oslo, Norway
• c.a.drevon_at_medisin.uio.no
• http//folk.uio.no/christia

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Disposition

• Definition of lipids
• Lipidomics
• Classes of lipids
• Analyses
• Application of knowledge to life health
• Integrated metabolism
• Literature

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Lipidomics - What how much?

• Lipids are insoluble in water soluble in
  organic solvents like ether, methanol
  chloroform
• Heterogenous group
• Constitute a large fraction of the body (3 - 70
  ) as well as the diet ( 30 15 E )

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Lipidomics - subgroup of metabolomics

• Metabolomics - includes quantitative and
  comprehensive analysis of all metabolites
• Lipidomics - quantification and characterization
  of all lipids in cells/organisms to determine the
  molecular mechanisms by which they operate
• Modified after Hahn Gross, J Lipid Res 2003,
  44, 1071-9

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Lipid classification Fahy et al, JLR 2005, 46,
839-61

• Fatty acyls (FA)
• Glycerolipids (GL eg MAG, DAG, TAG)
• Glycerophospholipids (GP eg PC, PI, PE, PS)
• Sphingolipids (SL)
• Sterol lipids (ST eg cholesterol, bile acids,
  estrogens, vitamin D)
• Prenol lipids (PR eg farnesol, vitamin E K)
• Saccharolipids (SL eg UDP-3-O-(3R-OH-tetradecanoyl
  )-aD-N-acetylglucosamine)
• Polyketides (PK eg afflatoxin B1)

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Some fatty acids
?-nomenclature Methyl-end
Carboxyl-end Saturation
Stearic 180
COOH Saturate
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Oleic 181?-9
COOH Monoene
Linoleic 182?-6
COOH PUFA
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3

?-Linolenic 183? 3
COOH PUFA
3
EPA 205n-3
COOH PUFA
3
DHA 226n-3
COOH PUFA
EPA eicosapentaenoic acid
DHAdocosahexaenoic acid

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Straight-chain fatty acids FA0101 Methyl
branched fatty acids FA0102 Unsaturated fatty
acids FA0103 Hydroperoxy fatty acids
FA0104 Hydroxy fatty acids FA0105 Oxo fatty
acids FA0106 Epoxy fatty acids FA0107 Methoxy
fatty acids FA0108 Halogenated fatty acids
FA0109 Amino fatty acids FA0110 Cyano fatty
acids FA0111 Nitro fatty acids FA0112 Thia
fatty acids FA0113 Carbocyclic fatty acids
FA0114 Heterocyclic fatty acids
FA0115 Mycolic acids FA0116 Dicarboxylic
acids FA0117
Fatty acids conjugates FA01
10
Octadecanoids FA02 12-Oxophytodienoic acid
metabolites FA0201 Jasmonic acids
FA0202 Eicosanoids FA03 Prostaglandins
FA0301 Leukotrienes FA0302 Thromboxanes
FA0303 Lipoxins FA0304 Hydroxyeicosatrienoic
acids FA0305 Hydroxyeicosatetraenoic acids
FA0306 Hydroxyeicosapentaenoic acids
FA0307 Epoxyeicosatrienoic acids
FA0308 Hepoxilins FA0309 Levuglandins
FA0310 Isoprostanes FA0311 Clavulones
FA0312 Docosanoids FA04 Fatty alcohols
FA05 Fatty aldehydes FA06 Fatty esters
FA07 Wax monoesters FA0701 Wax diesters
FA0702 Cyano esters FA0703 Lactones
FA0704 Fatty acyl-CoAs FA0705 Fatty acyl-acyl
carrier proteins (ACPs) FA0706 Fatty acyl
carnitines FA0707 Fatty acyl adenylates
FA0708 Fatty amides FA08 Primary amides
FA0801 N-Acyl amides FA0802 Fatty acyl
homoserine lactones FA0803 N-Acyl ethanolamides
(endocannabinoids) FA0804 Fatty nitriles
FA09 Fatty ethers FA10 Hydrocarbons
FA11 Oxygenated hydrocarbons FA12 Other FA00
Fatty acyls FA classes and subclasses
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Some fatty acids
?-nomenclature Methyl-end
Carboxyl-end Saturation
Stearic 180
COOH Saturate
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Oleic 181?-9
COOH Monoene
Linoleic 182?-6
COOH PUFA
6
3

?-Linolenic 183? 3
COOH PUFA
3
EPA 205n-3
COOH PUFA
3
DHA 226n-3
COOH PUFA
EPA eicosapentaenoic acid
DHAdocosahexaenoic acid

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Fatty acids conjugates Wenk, Nature Rev 2005,
4, 594-610
Lipid ID
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Bovine milk fatty acids (Taugbøl et al 2005)
GC-FID 200 m CP SIL-88 Hydrogen
181c9
60
100
180
120
80
183c9.12.15
182c9.12
205
204
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18 trans fatty acids in milk (Taugbøl et al 2005)
C181 c9
C181 c11
C181 t11
C181 t10
C181 t6-8
C181 t9
C181 c13
C181 t5
C181 t4
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Mechanisms of action for fatty acids
Platelets
W blood cells
Chemotactic agents


n-3
n-3
1) Precursors for eicosanoids

2) Substrate for enzymes
COOH
CH3
COOH
CH3


3) Peroxidation
Red blood cells


4) Membrane flexibility
more flexible



5) Acylation of proteins
FA
Protein
membrane

FA


6) Transcription factors
Nucleus
DNA
Promoter
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Biological effects of eicosanoids
Fatty acid AA EPA AA EPA AA EPA Enzyme Cell
type Eicosanoids TXA2 TXA3 PGI2 PGI3 LTB4 LTB
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Lipoxigenase
Cyclooxigenase
Endotelial cells
Platelets
Leucocytes
BIOLOGIC EFFECT Aggregation
Antiaggregation Vasoconstriction
Vasodilatation Chemotaxis

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Food sources of fatty acids

• FATTY ACID C-ATOMSDOUBLE BONDS SOURCES
  
• Saturates 12-16 Milk, meat, hard
• trans fat margarines, cakes, bisquits
• Linoleic acid 182, n-6 Vegetable oils, grain
• ?-linolenic 183, n-3 Green leaves,
  linseed, soybean canola oil
• EPA 205, n-3 Marine animals, cod
  liver fish oil
• DHA 226, n-3
  Marine animals, cod liver
  fish oil

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EFA deficiency in man n-6 n-3

• Large stores of PUFA in adipose tissue
• Extensive bowel resection
• Long term parenteral feeding
• 182, n-6 in plasma
• Appearance of 203, n-9 in plasma
• Scaling of epidermis
• High plasma cholesterol

• Scaly haemorrhagic dermatitis
• Haemorrhagic folliculitis of the scalp
• wound healing
• growth in children
• visual acuity
• Neuropathy
• conc of n-3 fatty acids in tissue lipids

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Fettsyrer plasma kolesterol (Müller et al.
Lipids 2001, 36, 783-791)
?kolesterol mM 120 0.01 140
0.12 160 0.057 TransMarin 0.039 TransVeg
0.031 181 - 0.0044 182/3 - 0.017
?LDL-kolesterol mM 0.01 0.071 0.047
0.043 0.025 - 0.0044 - 0.017
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Glycerolipids
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Glycerphospholipids
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Sterols
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Sterol lipids (ST) classes subclassesFahy et
al, JLR 2005, 46, 839-61
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Sphingolipids
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Prenyl lipids (PL) classes subclasses
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Vitamin K structure
Phylloquinone K1, in plants
R
R
3
Menaquinone K2, in bacteria
R
4-13
Menadione K3, synthetic
R 0
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Vitamin K derivatives

• Includes a 2-metyl-1,4-naftokinone (menadione)
  ring, with different isoprenyl-groups in
  3-position
• In plants - a 20-carbon-phytylgroup bound to
  menadione (phylloquinone K1)
• In bacteria - polyisoprenyl-sidechains with 4-13
  isoprenyl-units to (menaquinone K2)
• Unsaturated isoprenyl-sidechains w/variable
  length (MK-n)
• All possible combinations represent 70-80
  derivatives

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From lipids to DNA or opposite?
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THE CHROMATOGRAPHIC PROCESS - PARTITIONING
MOBILE PHASE (gas or liquid)
Detection
Sample injection
STATIONARY PHASE (solid or heavy liquid on a
support system)
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Mass spectrometry (MS)
Matrix Assisted Laser Desorption- Ionization (or
MALDI) electrospray ionization (ESI)
_at_Quadrupoles, time-of-flight (TOF) tubes,
cylindrical and linear ion traps, ion cyclotron
resonance cells, magnetic/electric sectors
Analyser
Detector
Source
MS detectors include channel electron
multipliers (CEM), and multi channel plates (MCPs)
Injection
Further details http//www.astbury.leeds.ac.uk/Fa
cil/MStut/mstutorial.htm
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Tandem mass spectrometry (MS)
Includes two MSs in series connected by a
collision cell that can fragment molecules. A
sample is sorted and weighed in the first MS,
then broken into pieces in the collision cell,
and sorted and weighed in the second MS
2 analysers
Detector
Source
Injection
Matrix Assisted Laser Desorption-Ionization (or
MALDI) electrospray ionization (ESI)
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Analytical tools in lipdomics
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Analytical tools in lipdomics II
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Living cells in lipdomics
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Lipidomics in nutrition research
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Cellular lipidomics Van Meer, EMBO Journal 2005,
24, 31593165
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Cellular lipidomics Van Meer, EMBO Journal 2005,
24, 31593165
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Cellular lipidomics Van Meer, EMBO Journal 2005,
24, 31593165
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Cellular lipidomics Van Meer, EMBO Journal 2005,
24, 31593165
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Toward the implementation of metabolomic
assessments of human health nutrition Watkins
German, Current Opinion in Biotechnology 2002,
13512516
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Computational prediction of human metabolic
pathways from the complete human genome Romero P
et al. Genome Biology, 2004, 6, R2

• BACKGROUND
• A computational pathway analysis of the human
  genome that assigns
• enzymes encoded therein to predicted metabolic
  pathways
• Pathway assignments place genes in their larger
  biological context, and are the necessary first
  steps toward quantitative modeling of metabolism

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Computational prediction of human metabolic
pathways from the complete human genome Romero P
et al. Genome Biology, 2004, 6, R2

• RESULTS
• Assigns 2,709 human enzymes to 896 bioreactions
• 622 enzymes are assigned roles in 135 predicted
  metabolic pathways
• Predicted pathways closely match known
  nutritional requirements
• Identified probable omissions in the human
  genome annotation as 203 pathway holes (missing
  enzymes in the predicted pathways)
• Identified putative genes to fill 25 of the
  holes
• The predicted human metabolic map is described
  by a Pathway/ Genome Database called HumanCyc, at
  http//HumanCyc.org
• Compared the predicted human metabolic pathway
  complement to the pathways of Escherichia coli
  and Arabidopsis thaliana, and identified 35
  shared pathways among all three organisms

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Computational prediction of human metabolic
pathways from the complete human genome Romero P
et al. Genome Biology, 2004, 6, R2
Curated HumanCyc pathway for oxidative ethanol
degradation. This pathway was not predicted by
PathoLogic, but was entered into HumanCyc as part
of our subsequent literature curation effort. The
flask icon in the upper-right corner indicates
this pathway is supported by experimental
evidence.
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Computational prediction of human metabolic
pathways from the complete human genome Romero P
et al. Genome Biology, 2004, 6, R2

• CONCLUSION
• Elucidates a significant portion of the human
  metabolic map
• Indicates probable unidentified genes in the
  genome
• HumanCyc provides a genome-based view of human
  nutrition associating essential dietary
  requirements with metabolic pathways supported by
  the human genome
• The database places many human genes in a
  pathway context, thereby facilitating analysis of
  gene expression, proteomics, and metabolomics
  datasets via a publicly available online tool
  (Omics Viewer)

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• Synthesis of malonyl-CoA is the first committed
  step of FA synthesis
• Acetyl-CoA carboxylase (ACC) is the major site
  of regulation of fatty acid synthesis
• Citrate stimultes ACC
• Palmitoyl-CoA inhibits ACC

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Integrated energy metabolism

• Food enters the body as a mixture of lipids,
  carbohydrates proteins
• These compounds are digested, assimilated and
  stored as triacylglycerol glycogen, mainly in
  adipose tissue, liver muscle
• gt 90 of total energy reserves are TAG
• Glycogen reserves are only used when ATP must be
  produced very quickly or when energy is scarce

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Integrated energy metabolism

• All cells can produce ATP from CH in the absence
  of 02 (glycolysis) in the cytoplasm
• NAD can be reduced to NADH used for ATP
  synthesis
• NAD is regenerated by oxidizing NADH converting
  pyruvate to lactate
• Anaerobic glycolysis can
• produce ATP very quickly
• without oxygen
• Anaerobic glycolysis is inefficient (1/18 of
  mitochondrial ox)
• When ATP is not rapidly required, and when O2 is
  present in sufficient amounts, pyruvate is fed
  directly into the Krebs cycle

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Integrated energy metabolism

• ATP is efficiently produced via oxidation with
  O2 in mitochondria
• Fatty acids are metabolised to acetyl-CoA by
  mit/perox b-oxidation
• Glycolysis catabolizes glycogen to pyruvate
  before conversion to acetyl-CoA
• Proteins are converted to individual amino acids
  that enter oxidation pathways as pyruvate,
  acetyl-CoA or directly as intermediates of the
  Krebs cycle
• b-oxidation, glycolysis and the Krebs cycle
  produce energy-rich NADH FADH
• Synthesis of ATP is linked to oxidation by O2 to
  NAD and FAD
• The pathway transfering energy from NADH FADH
  to ATP (respiratory chain) is located to the
  inner mitochondrial membrane

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Lipoproteiner
Kolesterol ester
Apoprotein
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Lipoproteiner
Næringsstoffer
Gallesyrer, kolesterol
LDL
Makrofager etc
LDL
KMR
LDL
VLDL
KM
IDL
Fibroblaster etc
LPL
LPL
HDL
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Lipoproteins Genetic Disorders Anne K Soutar
ELS, 2002 Macmillan Publishers Ltd, Nature
Publishing Group, www.els.net
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Lipoproteins Genetic Disorders Anne K Soutar
ELS, 2002 Macmillan Publishers Ltd, Nature
Publishing Group /, www.els.net
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Literature sources

• http//folk.uio.no/christia/
• Drevon, Blomhoff Bjørneboe Mat Medisin,
  2007
• Drevon CA Saarem K Omega-3 fatty acids
  metabolism and mechanisms of action of essential
  fatty acids. Peter Möller, 2005, pp 1-34
• Lipoproteins Genetic Disorders Anne K Soutar
  ELS, 2002 Macmillan Publishers Ltd, Nature
  Publishing Group, http//www.els.net
• The medical biochemistry page http//www.indstate
  .edu/thcme/mwking/home.html
• Romero P et al. Genome Biology, 2004, 6, R2
• Fahy et al, JLR 2005, 46, 839-61