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TRANSCRIPT
An Introduction to Metabolomics
Soumen Kanti Manna
Laboratory of Metabolism Center for Cancer Research
NCI, NIH
Reich, K. et al J. Invest. Dermatol. (2011)
Psoriasis # 1
Psoriasis # 2
Before After IL-10
Chemical Hub
Chemical Stimuli
Electromagnetic Radiations Electric/Magnetic fields
(Stellar bodies)
Biochemicals (Other life forms) Geochemicals
Synthetic chemicals
Chemical Stimuli
Chemical Response
Electromagnetic Radiations Electric/Magnetic fields
(Stellar bodies)
Biochemicals (Other life forms) Geochemicals
Synthetic chemicals
Change in Chemical Composition
Chemical Hub
Inside the Reactor Interplay between the ‘-omes’ : Metabolome is the outcome
Goodacre, R. Metabolomics (2005)
Phenotype
What may happen
What appears to be happening
What makes them happen
What actually happening or will happen
Where to Start?
What is Metabolomics?
Global analysis of metabolites (biochemicals)
Identification and measurement of metabolites in a cell/tissue/bio-fluid
Characterization of the effect of endogenous and exogenous perturbation on metabolite composition and networks
Elucidation underlying biological processes
Novel diagnostic/prognostic/therapeutic tool
What Can Be Probed?
Any system of metabolic networks Any process that affects with metabolism
Any matrix comprised of metabolites
Subject to the availability of reliable and sensitive high-throughput techniques!
Metabolomics Techniques
NMR Spectroscopy:
Positive: Oldest, reliable, direct information on chemical structure
Negative: Low sensitivity (inherent) and resolution
Mass Spectrometry:
Positive: High sensitivity and resolution
Negative: Indirect structural information and platform variability
Capillary Electrophoresis:
Positive: High sensitivity
Negative: Indirect structural information and not suitable for non-polars
Analytical Platforms
Detection limit
Advantages Disadvantages
NMR > 10-6 M Min sample prep Structural info
Sensitivity Spectral interpretation
Mass Spectrometry > 10-18 M Sensitivity Molecular diversity
Indirect structural info Sample prep
Capillary electrophoresis
> 10-15 M Sensitivity Indirect structural info Molecular diversity Sample prep
Urine (Non-invasive
sampling)
MS/MSPossible chemical formula
List of compounds
Confirmation
Quantitation
Chromatogram
Data annotation
Multivariate statistics
UPLC-ESIMS / GC-EIMS
List of ions
Informed guess
(Synthesis/derivatization/deconjugation)
Pathway analysis
Typical Workflow
Healthy Control/wild-typePatient/Treated/Mutant
Urine, Serum, feces, bile, CSF
Vehicle
Treated
3 D Opportunities
Deeper (biochemical) phenotyping.
Decoding signatures of biomolecular events at systems level.
Development of novel diagnostic/prognostic tools as well as
therapeutic strategies through generation, examination and
validation of functional hypothesis.
Instruments: A. Global Analysis:
UPLC-(ESI)QTOFMS Premier (Waters)
UPLC-(ESI)QTOFMS SYNAPT HDMS (Waters)
UPLC-(ESI)QTOFMS XEVO G2 (Waters)
GC-MS (Agilent)
GCxGC-MS PEGASUS 4D (LECO)
B. Targeted Analysis:
UPLC-(ESI)TQMS-XEVO (Waters)
Metabolomics at NCI/LM/NAS
Technique: Hyphenated Mass Spectrometry
Metabolomics at NCI/LM/NAS Disease and Disorders:
Characterization of metabolic imprint of pathological processes.
Identification of metabolic biomarker of diseases.
Liver disease, obesity, diabetes, kidney damage, cancer (Lung, GI, Leukemia).
Radiation Exposure:
Identification of noninvasive radiation exposure biomarkers.
Effect of ageing, history of exposure and genetic polymorphism.
Xenobitic Exposure:
Pharmacometabolomics of drugs and dietary supplements.
Effects of exposure and toxicity on endogenous metabolism.
Effect of dietary supplements on carcinogenesis.
Gene Function:
Role of genes in metabolism using genetically modified/transgenic animals, tissues,
cell lines along with other ‘-omics’ platforms and molecular biology.
Coordinate Chemical Interaction
In Search of High-throughput Early Noninvasive
Biomarker for Alcoholic Liver Disease
Alcoholism: A Global Challenge
Alcoholism is a common and increasing problem worldwide!
The tendency to develop alcoholism-related complications is increasing in
developing countries.
WHO status report (2011) attributed more deaths to alcoholism than those to
HIV/AIDS, violence or tuberculosis.1
A significant portion of alcohol-related deaths are caused by alcoholic liver
disease.2
1 http://www.who.int/mediacentre/news/releases/2011/alcohol_20110211/en/index.html 2 Hoyert, et al Natl Vital Stat Rep (2006); Welte, et al J Study Alcohol (2001)
Alcoholic Liver Disease (ALD)
Liver Cancer
Prevention is Better Than Cure!
Asymptomatic
Reversible
Liver Cancer
At early stages ALD is reversible but asymptomatic.
The development and progression of the disease varies significantly.
Regular screening of alcoholics can prevent fatal consequences.
What are the methods of diagnosis?
ALT, AST, GGT
Non-specific
Liver biopsy
Invasive, complications
Patient history along with other clinical symptoms
Unwillingness of patients to reveal drinking habit
Asymptomatic at early stages
Diagnosis and Challenges
Noninvasive, etiology-specific tool required for screening and diagnosis.
Metabolomics: An alternative ??
Considerable differences in alcohol metabolism, physiological response and
ALD susceptibility between as well as within ethnic groups.
The severity of the disease and mortality depends of genetic, environmental
factors as well as existing liver diseases.
Epidemiological Variation
Mandayam et al, Semin. Liv. Dis. (2004) Day and Bassendine, Gut (1992) Douds et al, Alcohol and Alcoholism (2003)
Noninvasive genetic background-independent biomarkers for
early stages of ALD are highly warranted!!
To find early noninvasive genetic background-independent high-
throughput biomarker/s for ALD.
To understand the biochemistry underlying ALD pathogenesis.
Aim & Motivation
Background
Fat deposition is the earliest observable change in ALD.
Peroxisome proliferator receptor alpha (PPARα) is a key regulator of lipid
metabolism.1
Alcohol-fed Ppara-null mice develops liver pathology very similar to early stages
human ALD.2
129S4/SvJ (129S) and C57BL/6N (BL6) mice have different genetic
backgrounds.
These mice differ considerably with respect to biochemical response to and
outcome of xenobiotic insult.3
1 Lee et al Mol. Cell. Biol. (1995) 2 Nakajima et al Hepatology (2004) 3 Syn et al, Liver Int. (2009); Liu et al, Toxicol. Appl. Pharmacol (2001)
Experimental Design
Control
4% EtOH
Chromatogram
MassLynx
SIMCA-P+
UPLC-QTOF
Male 129S WT and Ppara-null mice
Biomarker Identification
and Quantitation
Repetition and validation in BL6
Histology
↓ Model Validation
Traditional Biochemistry vs Metabolomic Signature
Metabolomic signature can distinguish animals undergoing
ALD pathogenesis!
Manna et al J. Proteome Res. (2010)
0
125
250
ALT
(U/l)
WTControl
Ppara-nullControl
WTAlcohol
Ppara-nullAlcohol
Affected Metabolic Pathways
Using MassTRIX server 1
(considering H+, Na+-adducts and mass error < 5ppm)
1 Suhre, K. and Schmitt-Kopplin, P. Nucl Acid Res., 2008
ALD-sensitive changes in tryptophan metabolism!!
Manna et al J. Proteome Res. (2010)
0 1 2 3 4 5 60
8
16
Months
Indo
le-3
-lact
ic a
cid
(µM
/mM
Cre
atin
ine)
0 1 2 3 4 5 60
8
16
Months
Indo
le-3
-lact
ic a
cid
(µM
/mM
Cre
atin
ine)
*
* # * #
* # * #
ALD-specific metabolic signature
control
Alcohol
Exclusive elevation of ILA and PLA in animals developind steatosis!!
WT Ppara-null
Manna et al J. Proteome Res. (2010)
Indo
le-3
-lact
ic a
cid
(µm
ol/m
mol
Cre
atin
ine)
Indo
le-3
-lact
ic a
cid
(µm
ol/m
mol
Cre
atin
ine)
Alcohol
Control
Biochemical Origin
C2H5OHEthanol NAD+
NADH + H+
SteatosisCH3CO2H
Acetic acid
NH
NH2
OOH
NH
O
OOH
Tryptophan
Ppara-null
CH3CHOAcetaldehyde
NH
OH
OOH
OHO
O
OHO
OH
Indole-3-pyruvic acid
Indole-3-lacticacid
Phenylpyruvicacid
Phenyllacticacid
OHO
NH2
EC 1.2.1.3
EC 1.1.1.2
Phenylalanine
ALD
Bio
mar
ker
EC 2.6.1.1 EC 1.4.3.2
EC 2.6.1.1 EC 2.6.1.5
EC 1.1.1.222
EC 1.1.1.110
Manna et al J. Proteome Res. (2011)
-50
0
50
-50 0 50
t[2]
t[1]
BL6 129Sv
PCA, ESI+
ExposureLength
Traditional Biochemistry vs Metabolomic Signature
The overall metabolic fingerprints of BL6 and 129S mice are always different.
Is there any common signature of ALD pathogenesis??
Manna et al J. Proteome Res. (2011)
NH
NH2
OOH
OHO
NH2
NAD+
NADH + H+Steatosis
Alcohol
NH
OH
OOH
OHO
OH
Alcohol metabolism
Microbialmetabolism
Amino acidmetabolism
ω-oxidation
Alcohol metabolism
Microbialmetabolism
Amino acidmetabolism
ω-oxidation
B6 Ppara-null129S Ppara-null
Tryptophan Phenylalanine
Indole-3-lactic acid Phenyllactic acid
ALD-specific metabolic signature
Manna et al J. Proteome Res. (2011)
Significance
Noninvasive high-throughput diagnostic potential
Mechanistic elucidation.
Acknowledgements
LM, NCI, CCR
Dr. Frank J. Gonzalez
Mr. Kristopher Krausz
Dr. Andrew Patterson
Dr. Caroline Johnson
Dr. Fei Li
Dr. Zhang
Georgetown University
Dr. Albert Fornace Jr.
Dr.
Dr. Yan Cai
Mr. Jorge Paize
Thanks to all LM members!!
Remember
To be clear about why you want to do metabolomics.
To carefully design animal experiments with special attention to strain, gender, food,
habitat, body weight, etc.
To ensure you have knowledge of sample/data analysis as well as access to raw data.
To ensure that samples were randomized, QC samples run and look good and the
data was appropriately normalized.
Not to jump into any conclusion based on the accurate mass measurement.
To hold on to conclusion till metabolites are quantitated.
Not to assume that you had a complete coverage of the metabolome using a single
extraction protocol or analytical platform.
Not to forget that changes in biofluid can be caused by gut flora.
Thanks!!
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