Metabolomics

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 cs

Proteomics

Systems Biology Omics Transcriptomics

Metabolomics

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

25,000 Genes

Genomics

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 hippurate allantoin creatinine taurine hippurate

urea

creatinine citrate 2-oxoglutarate

water

succinate

fumarate

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 highthroughput 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 27hydroxylaseDeficiency 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 GuanidinoacetateMethyltransferase 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-HyperammonemiaHomocitrullinuria 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 Failure

TMAO, Dimethylamine, Urea, Creatinine (serum)

Kidney (Rat)

Renal Damage (chemical)

Acetone, Lactate, Ethanol, Succinate, TMAO, Dimethylamine, Taurine (urine & serum)

Kidney (Human)

Graft Dysfunction

TMAO, Dimetheylamine Lactate, Acetate, Succinate, Glycine, Alanine, (urine)

Kidney (Rat)

Graft Dysfunction Reperfusion Injury

TMAO, Citrate, Lactate, Dimetheylamine, Acetate (urine)

Kidney (Rat)

Reperfusion Injury (ischemia)

TMAO, Allantoin (serum)

Kidney (Human)

Graft Dysfunction CsA toxicity

TMAO, Alanine, Lactate, Citrate (urine & serum)

Kidney (Mouse)

Nephrectomy

Methionine, Citrulline, Arginine, Alanine (urine & serum)

Serine (serum)

Kidney (Mouse)

Nephrectomy

Guanidinosuccinate, Guanidine, Creatinine, Guanidinovalearate, (urine & serum)

Guanidinoacetate (urine)

Kidney (Human)

Acute Rejection

Citrate, Glucose, Urea Allantoin (urine & serum)

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