Metabolomics

Lab meeting , 8 0ctober 2009

Dr. M .Vishnu vardhan reddy

SGPGIMS ,Lucknow, India

 What is Systems Biology?
• Systems Biology - Systems biology is the
study of an organism, viewed as an integrated
and interacting network of genes, proteins
and biochemical reactions. The integration of
genomics, proteomics, metabolomics &
modeling
• The Goal: Predictive, Preventative and
Personalized Medicine -- Leroy Hood
 Genomics

MicroRnomi
Proteomics
cs

Systems
Biology -
Omics
Metabolomics Transcriptomics

Interactomics
Publications in –omics
 Genomics, Proteomics & Systems
Biology

Genomics

Proteomics

Systems Biology

1990 1995 2000 2005 2010 2015 2020

 The Pyramid of Life

Metabolomics
1400
Chemicals

Proteomics
2500 Enzymes

Genomics
25,000 Genes
Systems Biology is Multidisciplinary
 Applications of systems biology
• Basic Science/”Understanding Life”
• Predicting Phenotype from Genotype
• Understanding/Predicting Metabolism
• Understanding Cellular Networks
• Predicting Disease Outcome/Prognosis
• Understanding Pathogenicity/Toxicity
• Predicting Adverse Drug Reactions
• Improving Medical Efficiency/Efficacy
 Definitions
• Metabolomics
– Newly emerging field of 'omics' research
– Comprehensive and simultaneous systematic
determination of metabolite levels in the metabolome
and their changes over time as a consequence of stimuli
• Metabolome
– Refers to the complete set of small-molecule
metabolites
– Dynamic
 What is a Metabolite?
• Any organic molecule detectable in the body with a MW <
1000 Da
• Includes peptides, oligonucleotides, sugars, nucelosides,
organic acids, ketones, aldehydes, amines, amino acids, lipids,
steroids, alkaloids and drugs (xenobiotics)
• Includes human & microbial products
• Concentration > 1µM(limit of NMR detection & excludes
environmental pollutants)
What’s the Difference Between
Metabolomics and Traditional
Clinical Chemistry?

Throughput
(more metabolites, greater accuracy,
higher speed)
 Why is Metabolomics Relevant?
• Generate metabolic “signatures”
• Monitor/measure metabolite flux
• Monitor enzyme/pathway kinetics
• Assess/identify phenotypes
• Monitor gene/environment interactions
• Track effects from toxins/drugs/surgery
• Monitor consequences from gene KOs
• Identify functions of unknown genes
 Data gathering
• Separation Techniques
– Gas Chromatography (GC)
– Capillary Electrophoresis (CE)
– High Performance Liquid Chromatography (HPLC)
– Ultra Performance Liquid Chromatography (UPLC)
• Combination of Techniques
– GC-MS
– HPLC-MS
• Detection Techniques
– Nuclear Magnetic Resonance Spectroscopy (NMR)
– Mass Spectrometry (MS)
 Instruments in metabolomics

Computer
Cluster
NMR

MS
HPLC
Seperation Technique - GC
 Detection Technique - MS

• To identify and to
quantify metabolites
• Serves to both separate
and to detect
• Mass to charge ratios
• Using electron beam
• Ion source, mass
analyzer and detector
NMR Experiment

TMAO
creatinine
hippurate
allantoin creatinine taurine citrate

hippurate urea 2-oxoglutarate

water
fumarate succinate

ppm 7 6 5 4 3 2 1
 NMR versus MS
• Quantitative, fast
• Requires no work up or
separation
• Allows ID of 300+ cmpds at
once
• Good for CHO’s
• Not sensitive
• Needs MS or 2D NMR for
positive ID
• Very fast
• Very sensitive
• Allows analysis or ID of
3000+ cmpds at once
• Not quantitative
• Not good for CHOs
• Requires work-up
• Needs NMR for ID
 Metabolome analysis -four categories
• Metabolite Target Analysis-mainly used for screening
purpose- study of primary effects of any alteration, analysis can
be restricted to a particular metabolite or enzyme
• Metabolic Fingerprinting classifies samples according to their
biological relevance and origin and used for functional
genomics, plant breeding and various diagnostic purposes
• Metabolite Profiling- to study the number of compounds
belonging to a selected biochemical pathway
• Metabolomics to determine metabolic snapshots in a broad
and comprehensive way. In this, both sample preparation and
data acquisition are aimed at including all class of compounds,
with high recovery and experimental robustness and
reproducibility
 Bottlenecks in metabolomics
• Huge diversity of chemical structures and the large differences
in abundance-no single technology available to analyze the
entire metabolome
• Analytical variance- related to experimental approach
• Biological variance -arises from quantitative variation in
metabolite levels between plants of same species grown
under identical or as near as possible identical
• extract the information and interpret it in a biological context
from the vast amount of data produced by high-
throughput analyzers
How do we deal with data that don’t make biological
sense based on literature and common knowledge?
 Human Metabolome Project

• $7.5 million Genome Canada Project launched in Jan. 2005

• Mandate to quantify (normal and abnormal ranges) and identify

all metabolites in urine, CSF, plasma and WBC’s

• Make all data freely and electronically accessible

• Make all compounds publicly available (HML)

 Database for Metabolomics
• Biggest challenge of metabolomics is the current lack of appropriate database
and data exchange format

• MetaCyc -a database of experimentally elucidated metabolic pathways

comprising of about 700 pathways from more than 600 different organisms
• MetNet -information on networks of regulatory and metabolic interaction
in Arabidopsis
• Map Man - tool that displays large datasets on to diagrams of
metabolic pathways
• BioCyc

• BRENDA- represents the most comprehensive information system on the

enzymes and metabolic information The database contains data from atleast
83,000 different enzymes from 9800 different organisms
Human Metabolome Database

www.hmdb.ca
 The Human Metabolome Library

Repository of chemical samples for public redistribution

stored in -80°C freezers •309 metabolites in CSF,

•1122 metabolites in serum
•458 metabolites in urine
•300 metabolites in other tissues
and biofluids
 Metabolic Profiling: The Possibilities
• Toxicology Testing • Genetic Disease Tests
• Clinical Trial Testing • Nutritional Analysis
• Fermentation Monitoring • Clinical Blood Analysis
• Food & Beverage Tests • Clinical Urinalysis
• Nutraceutical Analysis • Cholesterol Testing
• Drug Phenotyping • Drug Compliance
• Water Quality Testing • Dialysis Monitoring
• Petrochemical Analysis • MRS and fMRI
 Main Applications

• Drug assessment
• Clinical toxicology
• Nutrigenomics
• Functional genomics
 Metabolic Profiling: The Possibilities
• Toxicology Testing • Genetic Disease Tests
• Clinical Trial Testing • Nutritional Analysis
• Fermentation Monitoring • Clinical Blood Analysis
• Food & Beverage Tests • Clinical Urinalysis
• Nutraceutical Analysis • Cholesterol Testing
• Drug Phenotyping • Drug Compliance
• Water Quality Testing • Dialysis Monitoring
• Organ Transplantation • MRS and fMRI
 Medical Metabolomics
• Generate metabolic “signatures” for disease states or host responses

• Obtain a more “holistic” view of metabolism (and treatment)

• Accelerate assessment & diagnosis

• More rapidly and accurately (and cheaply) assess/identify disease

phenotypes

• Monitor gene/environment interactions

• Rapidly track effects from drugs/surgery

 140+ Detectable Conditions
• Adenine
Phosphoribosyltransferase
Deficency
• Adenylosuccinase Deficiency
• Alcaptonuria
• α-Aminoadipic Aciduria
• β-Aminoisobutyric Aciduria
• α-Aminoketoadipic Aciduria
• Anorexia Nervosa
• Argininemia
• Argininosuccinic Aciduria
• Aspartylglycosaminuria
• Asphyxia
• Biopterin Disorders
• Biotin-responsive Multiple
Carboxylase Deficiency
• Canavan’s Disease
• Carcinoid Syndrome
• Carnosinemia
• Cerebrotendinous
Xanthomatosis/sterol 27-
hydroxylaseDeficiency
• Citrullinemia
• Cystathioninemia
• Cystinosis
• Cystinuria (Hypercystinuria)
• Diabetes
• Dibasic Aminoaciduria
• Dicarboxylic Aminoaciduria
• Dichloromethane Ingestion
• Dihydrolipoyl Dehydrogenase
Deficiency
• Dihydropyrimidine Dehydrogenase
Deficiency
• Dimethylglycine Dehydrogenase
Deficiency
• Essential Fructosuria
• Ethanolaminosis
• Ethylmalonic Aciduria
• Familial Iminoglycinuria
• Fanconi’s Syndrome
• Folate Disorder
• Fructose Intolerance
• Fulminant Hepatitis
• Fumarase Deficiency
• Galactosemia
• Glucoglycinuria
• Glutaric Aciduria Types 1 & 2
• Glutathionuria
• Glyceroluria (GKD)
• D-Glyceric Aciduria
• Guanidinoacetate-
Methyltransferase Deficiency
• Hartnup Disorder
• Hawkinsinuria
• Histidinemia
• Histidinuria
• Homocystinsufonuria
• Homocystinuria
• 4-Hydroxybutyric Aciduria
• 2-Hydroxyglutaric Aciduria
• Hydroxykynureninuria
• Hydroxylysinemia
• Hydroxylysinuria
• 3-Hydroxy-3-methylglutaric Aciduria
• 3-Hydroxy-3-methylglutaryl-Co A Lyase
Deficiency
• Hydroxyprolinemia
• Hyperalaninemia
• Hyperargininemia (Argininemia)
• Hyperglycinuria
• Hyperleucine-Isoleucinemia
• Hyperlysinemia
• Hyperornithinemia
• Hyperornithinemia-Hyperammonemia-
Homocitrullinuria Syndrome (HHH)
• Hyperoxaluria Types I & 2
• Hyperphenylalaninemia
• Hyperprolinemia
• Hyperthreoninemia
 Why Metabolomics For Transplants?
• Relatively non-invasive (no need for biopsy, just
collect urine, blood or bile)
• Can be quite organ specific
• Very fast (<60 s for an answer) & cheap
• Metabolic changes happen in seconds, gene,
protein and tissue changes happen in minutes,
hours or days
• Allows easy longitudinal monitoring of patient
(or organ) function (pre&post op)
 Applications In Transplantation
Organ Condition Metabolite(s) Increased Metabolite(s) Decreased

Kidney (Human) Chronic Renal TMAO, Dimethylamine, Urea,

Failure Creatinine (serum)

Kidney (Rat) Renal Damage Acetone, Lactate, Ethanol, Citrate, Glucose, Urea
(chemical) Succinate, TMAO, Allantoin (urine & serum)
Dimethylamine, Taurine
(urine & serum)
Kidney (Human) Graft Dysfunction TMAO, Dimetheylamine
Lactate, Acetate, Succinate,
Glycine, Alanine, (urine)

Kidney (Rat) Graft Dysfunction TMAO, Citrate, Lactate,

Reperfusion Injury Dimetheylamine, Acetate (urine)

Kidney (Rat) Reperfusion Injury TMAO, Allantoin (serum)

(ischemia)

Kidney (Human) Graft Dysfunction TMAO, Alanine, Lactate,

CsA toxicity Citrate (urine & serum)

Kidney (Mouse) Nephrectomy Methionine, Citrulline, Arginine, Serine

Alanine (urine & serum) (serum)
Kidney (Mouse) Nephrectomy Guanidinosuccinate, Guanidinoacetate (urine)
Guanidine, Creatinine,
Guanidinovalearate,
(urine & serum)
Kidney (Human) Acute Rejection Nitrates, Nitrites, Nitric oxide
metabolites (urine)
 Future challenges

• Database

• Standardisation

• Diversity/variation of metabolomic data

– More efficient ways of identification

• Better models for interpretation of data

• Integration with other 'OMICS'

 Concluding Comments
• Metabolomics is rapidly becoming the “new clinical chemistry”
• Metabolomics complements genomics, proteomics and
histology
• Metabolomics allows probing of rapid physiological changes or
events that are not as easily detected by microarrays or
histological methods
• metabolomics offers a unique opportunity to look at
genotype-phenotype as well as genotype-envirotype
relationships
• a key limitation to metabolomics is the fact that the human
metabolome is not at all well characterized.
Going From Networks in vivo to
Networks in silico