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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