NMR Spectroscopy Based Metabolomics and
Its Multidisciplinary Research Applications
G. A. NAGANA GOWDA
ANALYTICAL CHEMISTRY DIVISION
West Lafayette, IN--47907
Genes
Metabolic Profiling
mRNA enzymes small molecules (Metabolites)
Causes of
metabolic
disturbance
In this era of omic scices,
•Genetic defects
•mRNA defects
•Enzyme defects
•Drug effects
Modest change in enzyme activity > substantial changes in metabolites
What are Metabolites?
•
•
•
•
Any organic molecule detectable in a biological
system with a MW < 1000 Da
Includes peptides, oligonucleotides, sugars,
nucleosides, organic acids, ketones, amines,
amino acids, lipids, steroids, alkaloids and drugs
Includes both human & microbial products
Concentration > 1μM
Metebolomics/metabonomics/ metabolic profiling
Metabolomics also commonly known as metabonomics or
metabolic profiling deals with detection and quantitative
analyses of hundreds or thousands of metabolites from
biological samples in a single step.
Biological samples may be from
Human
Animal
Plant
Food
Potential Metabolomics Applications
• Early disease diagnosis and prognosis
• To determine whether a treatment is working or not
• Monitor healthy people to detect early signs of disease: Predicting the
risk of disease
• Clinical Chemistry of 21st Century!
•
•
Drug development: To speed the discovery and development of drugs
Predict potentially harmful effects of drugs (toxicity)
•
•
•
Identification of perturbed metabolic pathways in disease
Discovering new functions of gene
When combined with genomics, transcriptomics and proteomics analysis,
metabolomics offers potential to understand the whole biological system.
• Food and Nutrition
• investigations of plants
• Animal studies
Personalized Medicine
Individual’s health or disease profile from body fluid sample
One of the goals of metabolomics is to
personalize the medicine
Finger print
Urine NMR
Urine samples
Metabolic finger print
Designing effective Treatment protocol based on individuals metabolic finger print
What are the advantages?
‰
Measures multiple (10’s to 100’s) metabolites
simultaneously
‰
No sample preparation or separation!!
‰
Allows metabolic profiles or “fingerprints” to be generated
‰
Highly quantitative (NMR)
‰
Can be automated
‰
High throughput < 60 s
Metabolomics requires powerful information rich analytical
technique to detect a high number of metabolites in one step
Non-selective
Sensitivity
Resolution
Reproducibility
Metabolite Identity
Quantitative
MS
NMR
Commonly used Analytical Techniques
Mass spectrometry (MS)
Nuclear magnetic resonance (NMR) spectroscopy
NMR Spectroscopy
•
Strengths of NMR
•
Detection of almost all the metabolites
•
Reproducibile
•
Quantitative
•
Non-invasive: Enables Repeated measurements
•
Only technique available for the analysis of intact
samples such as tissue, food, plants
•
Challenges
•
Resolution
•
Sensitivity
•
Metabolites identification in the complex spectrum
NMR
Facets of NMR
Spectroscopy- Lets you visualize molecular species and their 3D structures
inside the objects
Imaging- Lets you visualize organs and inside of the human body and brain
function
1D-Spectroscopy
2D Spectroscopy
3D Spectroscopy
ppm
-8.98
-9.00
-9.02
-9.04
-9.06
-9.08
-9.10
-9.12
-9.14
5.0
4.5
4.0
3.5
3.0
DOSY
2.5
2.0
1.5
ppm
Imaging
Functional MRI
NMR Spectroscopy
Metabolites are identified through atomic nuclei
•Metabolites are detected through
Hydrogen (H), Carbon (C), Nitrogen (N) or Phosphorus (P)
•1H- almost all compounds in biological samples contain hydrogen atoms
High Natural abundance (99.98%)
High sensitivity
Commonly used
•13C-almost all compounds in biological samples contain carbon atoms
Low natural abundance (1.1%)
Low sensitivity
•15N-many compounds in biological samples contain nitrogen atoms
Very low natural abundance (0.37%)
Very low sensitivity
•31P-many compounds in biological samples contain nitrogen atoms
100% Natural abundance
High sensitivity
1H
Water molecule
Absence of magnetic field
NMR spectrum
Tiny magnets
Inside the magnet
water
Magnetic field
Random orientation
Observable part
Magnetic field
z
ALIGNED along or Opposite
to the applied magnetic Field
x
z
z
Difference
Observable part
Mz
Lower magnetic field
y
x
z
x
Mz
z
Observable part
y
x
x
Higher magnetic field
NMR Signal
Magnetic field
z
z
Mz
My
y
x
RF pulse
(6 μs)
y
x
z
water
y
NMR Signal
x
FT
FID
f
Frequency > identity
of the metabolite
r
Intensity > concentration
of the metabolite
e
How do you get thousands of NMR signals for bodyfluids?
z
z
Mz
My
y
x
RF
y
Urine
z
6 μs
y
Urine 1H NMR spectrum
x
FID
FT
Biosamples
(Serum, Urine, Tissue, etc.)
Metabolomics: Flow diagram
Sample preparation
(Extraction, derivatization, buffering for NMR, GC-MS, etc.)
1D/2D NMR
(1D 1H NOESY, 2D TOCSY,
HRMAS, etc.)
10 8
6 4
ppm
2
1D/2D MS
(LC-MS, GC-MS,
DESI-MS, etc.)
100
0
200
300
400
m/z
Data preprocessing
(Baseline correction, binning, scaling, normalization, etc.)
Unsupervised/supervised statistical analysis
(PCA, PLS-DA, O-PLS, etc.)
Scores
PC2
Loadings
Disease
Control
5
4
3
2
PC1
Putative Biomarkers
(Identity, quantity)
Validation and Biological Understanding
Clinical Trails
1
0
Samples
¾
Body Fluids
Body fluids
¾
¾
Cell Cultures
¾
Serum/plasma
¾
Tissue
¾
Urine
¾
Perfused organs
¾
Saliva
¾
small animals
¾
Bile
¾
Humans
Food
fruit, meat, fish,
cheese, pasta
¾
Plants
Samples from animals or humans
Urine samples
• Collect in plain tubes on ice or in a refrigerator
• Use 0.05 to 0.1 % wt/vol NaN3
• Refrigerate frozen at -40oC until analyzed by NMR
Blood
• Plasma: Collect in lithium heparin to get plasma
(not in EDTA or citrate)
• Serum: Collect in plain tube (no additive) leave to
coagulate for about 30 min. Centrifuge at 1600 g for 15
min at 4o C to separate blood cells
• Store the supernatant at -40oC until analyzed by NMR
Samples from animals or humans
Tissue
Freeze the tissue samples rapidly in liquid nitrogen (~100mg)
to arrest biochemical reactions
Store the samples at -80oC
Phosphate Buffer (pH = 7.4)
Na2HPO4
28.85 g
NaH2PO4
5.25 g
NaN3
3mM
Fill in 1 liter flask, make up with water and
dissolve the contents
Sample preparation for NMR experiments
Urine
• 300 μl urine and 300 μl of phosphate buffer (pH=7.4)
•
•
Centrifuge and remove particulate matter.
550 μl in 5 mm NMR tube; Insert a coaxial tube filled with D2O+TSP
Serum/plasma
500 μl and 50 μl phosphate buffer
Tissue
Intact Tissue: High Resolution NMR with Magic angle sample spinning
or
Extract polar and lipid metabolites
5mm NMR tube
Nature Protocols (2007) 2(11), 2692-2703
Is the sample amount fixed?
• NO
• If the available sample quantity is less, you can use less
• Need to use appropriate NMR accessories
• As little as 5 μl can be used with micro-coil NMR probes
Untreated Serum/
urine
Superconducting magnet
Higher sensitivity by reducing the temperature of the NMR coil and preamplifier
DRX500 with Cryo-probe at Purdue
Higher sensitivity and resolution by increasing the magnetic field strength
Avance III 800 at Purdue
Temperature should be constant for the samples: 298K
Achieve good solvent (water) suppression
Normal 1D 1H NMR experiment- 1D NOESY presaturated
NMR experiments: Urine
•One dimensional NMR experiment
•There are several pulse techniques for 1D NMR
Residual
water
NOESY-presat
NMR experiments: serum/plasma
1d-NOESY-presat
Relaxation Edited
CPMG
Diffusion edited
NMR
High Resolution Magic Angle Spinning (HR-MAS NMR)
Samples such as tissue, food or plants
NMR is the method of choice
•
Additional interactions through space between
nuclear magnets (spins) which are orientation
dependent
•
Sample inhomogeneity leads differences in
magnetic susceptibility
•
Solution: magic angle sample spinning
•
Magic angle: 54.7o
•
•
•
•
•
•
B0
54.70
4 mm o.d. zirconium oxide rotor
Cut to a suitable size to fit into the rotor
Sample size ~ 10-15 mg,
Insert into the rotor with additional D2O
spun at the magic angle to Bo
spin rate ~ 4 kHz
Rotors (sample tubes)
High Resolution Magic Angle Spinning (HR-MAS NMR)
B0
54.70
•Biochemical changes can occur during NMR experiments
•Keep the sample cooled during NMR experiment
High Resolution Magic Angle Spinning NMR of tissue
400 MHz 1H CPMG MAS NMR spectra of cells and tissues (spin rate 4.2 kHz)
1D MAS NMR experiments require ~ 5 min
HRMAS NMR Spectra
15 mg tissue samples
Liver (cancer adjacent tissue)
Liver Cancer
J. Proteome Res. 6,2605, 2007
NMR of Extracts: Metabolic profiling of food
Fruit, meat, fish, cheese, pasta
•Soft cheese degradation in different packaging conditions by 1H-NMR
• Deals with the influence of packaging on the degradation of soft cheese
•
NMR spectra of aqueous cheese extracts were acquired as a function
of time during storage of cheese inside and outside the original package.
• 15 metabolites are quantified and used in a kinetic degradation model.
Magn. Reson. Chem. 2008, 46, 828–831
Chemometric analysis
Multivariate Statistical Analysis
10000
G-1
G-2
PC2
Disease
PC1
-8000
0
Control
-10000
NMR
PCA
8000
Chemometric analysis
Baseline correction
Data alignment
Chemometric analysis: Data pretreatment
•
Data binning: 32K to 4K:
-pH effects
•
Data Normalization: to compensate
-Dilution effects of urine
•
Data scaling
-equal weight for small and big
peaks
variance scaling: (division by the standard
deviation of the peak intensities across the set of
spectra) and
Parato scaling (division by the square root of
the standard deviations).
Approaches of Data analysis
ppm
7
6
5
4
3
2
Chemometric
(Pattern recognition)
Quantitative
Methods
25
TMAO
hippurate
allantoin creatinine taurine
PC2
20
creatinine
15
10
citrate
5
urea
hippurate
water
1
2-oxoglutarate
0
succinate
-5
fumarate
Diet1
-10
ppm
7
6
5
4
3
2
1
Diet3
-15
Diet2
-20
-25
-30
PC1
-20
-10
0
10
Quantitative
Chemometric
•
Metabolite Identification
No
•
Metabolite quantification
No
•
Handles wide range of
samples/conditions
Requires strict sample
uniformity
•
Allows identification of
diagnostic patterns
Allows identification of
diagnostic patterns
•
Use database for
identification
•
More NMR for identifying
new metabolites
Comparison of patients with different severity of coronary atherosclerosis
Predict the
occurrence and
severity of
coronary artery
disease using
blood plasma.
Brindle et al., Nature medicine 8, 2002.
Example: personalized medicine
‰ Administration of galactosamine hydrochloride (induce liver
disease) to a group of ten rats.
‰ Effects observed from NMR were variable enough that the rats
could be classified as either ‘responders’ or ‘non-responders’.
‰ PCA analysis showed some discrimination between responder and
non-responder groups in terms of their pre-dose metabolite
profiles.
PCA (predose)
Andrew et al., Nature letters 440, April 2006.
Metabolic Disorder: Aminoacylase I deficiency
Handbook of 1H-NMR
spectroscopy in inborn errors
of metabolism: body fluid
NMR spectroscopy and in
vivo
MR spectroscopy / von U.
Engelke, S. Moolenaar, S.
Hoenderop, E. Morava, M.
van der Graaf, A. Heerschap,
R. Wevers
Heilbronn: SPS
Verlagsgesellschaft, 2007
ISBN 3-936145-49-0
Some 2D NMR experiments for metabonomics
Experiment
Detected
nucleus
Indirect
axis
NMR interaction responsible
COSY
δH
δH
DQF-COSY
δH
δH
TOCSY
δH
δH
2J
NOESY
δH
δH
H-H Dipole rHH, conformation
ROESY
δH
δH
H-H Dipole rHH, conformation
HMQC
δH
δC
1J
CH C-H
HSQC
δH
δC
1J
CH C-H
HMBC
δH
δC
2J
CH
JRES (homo)
δH
JHH
Measurement of JHH
JRES (hetero)
δC
JCH
Measurement of JCH
2J
HH
2J
& 3JHH connectivity
HH
HH
& 3JHH connectivity
& 3JHH all in a spin system
& 3JCH C-C-H & C-C-C-H
Metabolites identification using 2D NMR
Double Quantum Filtered-COSY experiment
DQF-COSY
Jejunal aspirate
(fluid from small intestine)
More NMR experiments for identifying new metabolites: Total
Correlation Spectroscopy (TOCSY
-Rat kidney 17 mg
From Bruker
Identification of unknown metabolites using 2D NMR methods
HMBC
HSQC
DQF-COSY
SEFT
NMR metabolite Databases
¾
Several freely available electronic database
¾
Information on individual metabolite
¾
Human Metabolome Database (HMDB)
(http://www.hmdb.ca/)
¾
HMDB currently contains nearly 2500 metabolite
entries
Cryoprobe 13C NMR spectra
500 MHz
13C/1H
dual probe
Rat urine diluted 2:1 with buffer, 512 scans, 30 minute acquisition
Control
Dosed 90 mg/kg
hydrazine, 48 h
Lindon JC
Combining NMR and MS Methods:
Nuclear Magnetic
Resonance (NMR)
Spectroscopy
Sample
High resolution NMR
Mass Spectrometry
(MS)
New MS technologies
High-Throughput Screening by NMR
Metabolomics: Future Clinical chemistry tool?
Analytical Laboratory at LipoSciences
Assessment
of Risk of
coronary
heart disease
blood plasma
Turnover with
BEST-NMR
~ 150 sec
including
NMR-measurement
In vivo NMR 0.5, 1.0, 3.0,…….now it is 7 Tesla
Metabolites detection in vivo
Future Directions: Integration of these approaches
Improvements for NMR are needed for better sensitivity and resolution
z
Isotope tags
z
Cryoprobes
z
Micro-coil NMR
z
High magnetic fields
z
HRMAS
z
LC-NMR
z
LC-MS
z
GC-MS
Conclusions
•
Metabolomics is rapidly growing with its applications
spread in many areas of science: Biomedicine, Drug
discovery, Food and Nutrition, etc.
•
Metabolic analysis is a promising approach for the noninvasive monitoring of human health, by detecting early
stages of illness and applying the right treatment.
•
Metabolomics complements genomics, proteomics and
histology
•
Metabolomics allows probing of rapid physiological
changes or events that are not easily detected by
microarrays or histological methods
•
Metabolomics based personalized medicine will
eventually improve the health of the whole population.