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Developing standards for metabonomics as a clinical tool.
1. Developing standards for
metabonomics as a clinical tool.
Agnieszka M. Lichanska, Shaffinaz Abd Rahman
and Horst J. Schirra
School of Dentistry and IMB, University of Queensland, Australia
3. Genomics and proteomics tells you what
might happen, but metabolomics tells you
what actually did happen.
- Bill Lasley, University of California, Davis
5. Introduction
Metabonomics
•'The quantitative measurement of the dynamic multiparametric
metabolic response of living systems to pathophysiological stimuli or
genetic modification' (Nicholson et. al.)
•Use of statistical methods to detect changes in metabolites over
time or between groups.
Metabolomics
•The characterisation of all metabolites in a sample/organism.
… a lot of people use both terms interchangeably…
6. Metabolic changes
Genetic changes
(mutations)
Drugs, diet
Changes in metabolite
concentration
Blood Urine other biofluids
NMR, MS
spectra
Disease
Identification of
individual metabolites
Detailed analysis
Metabolic changes and their analysis
Metabolite
Database
7. Methods
• Bruker 500MHz spectrometer with sample
changer
• Urine from fasted male mice
– WT controls
– Mutant mice
– Age 2-12 months
• Samples frozen upon collection
• 1M Phosphate buffer
• TSP, D2O
8. Nuclear Magnetic Resonance (NMR) - based
metabonomics
• Advantages:
– Fully quantitative
– Non-destructive
– Minimal sample preparation
– High throughput
• What can be analyzed?
– Analysis of all types of biofluids (urine, plasma, saliva,
cerebrospinal fluid, sperm, synovial fluid, amniotic fluid)
– Solid samples (biopsies of organs and cell cultures)
9. Mouse studies - physiology of growth
hormone
WT 569 391 GHR -/-
Rowland, Lichanska et al 2005, MCB 25:66-77
Model - GHR KI mice
10. Mouse studies - physiology of growth
hormone
Schirra et al 2007, under review
11. Mouse studies - physiology of growth
hormone
Schirra et al 2007, under review
16. Methods
• Bruker 500MHz spectrometer with sample
changer
• Urine from a healthy volunteer
– 1st urine of the day
– Collected midstream
• 1M Phosphate buffer
• TSP, D2O
• Na Azide
• Storage conditions variable
17. Study design
Untreated Centrifuged
First morning
urine
Room
Temp.
on ice / -20°C
1% Sodium Azide Ultra-Filtrated
(MWCO 10 kDa)
The samples were stored either at -20o
C or at RT and were measured on
day 0, 2 and 9.
Sterile filtration
0.2µm
18. Day 0
1% Sodium Azide
Ultra-Filtrated Centrifuged
Untreated
Blue Spectra – Room Temperature
Red Spectra – In Ice
• Spectra are identical, irrespective of treatments
• Ultra-filtrated samples had addition glycerol signals
Glycerol
20. Comparison between treatments days 0-9
Day 0
Day 2
Day 9
Day 9
Day 2
Day 0
Centrifuged
Formate
Formate
Formate
Formate
Ethanol
Ethanol
Acetate
Acetate
Acetate
Acetate
Untreated
22. Summary
• Storing samples at RT caused them to have ageing effects
formed from microbial contamination in the samples
• Sterile filtration samples kept at -20 °C were the only
treatment that showed consistently no presence of those
metabolites
• Optimal method:
1. Sterile filter samples
2. add 1% sodium azide
3. Measure at Day 0
4. Store at -20°C
• Ethanol, acetate and formate signals should be excluded in
all statistical study as it was proven that elevated
concentrations of these metabolites were due to external
ageing reactions.
23. Metabolomics standards initiative (MSI,
http://msi-workgroups.sourceforge.net/ )
• Sample information
– Collection details (date, place, method, frequency)
– Extracts from tissues or solid state analysis - processing details
– Patient information (diet, drugs, infections, chronic diseases, etc)
– Volume collected, pH, osmolarity,
– Sample storage (temperature, additives)
– Sample processing details
• NMR/MS data acquisition
– QC procedures, used of internal standards,
– Instrument performance and maintenance logs has to be documented
– Acquisition protocol details - SOP establishment
– Instrument specifications
• Data analysis
– All data manipulations should be specified, MSI has a format for reporting
analysis
• Data format
– Exchangeable file format
– Raw data access for future re-analysis or reference
24. FDA and metabonomic studies
• Metabolomics data is currently being evaluated by the
voluntary genomics data submission (VGDS) group.
• Such data is likely to be included in FDA submissions to
support:
– A mechanism of a drug
– Metabolite/s are used biomarkers in evaluations
• Metabolic markers are seen as most relevant for
understanding the mechanism of action, defining the
safety of a compound, and for monitoring clinical
efficacy.
• A metabolomic report should include a number of
information mentioned before. The two important issues
are the selection of controls and the analysis methods
used to identify the biomarker/s
25. Acknowledgments
IMB
Dr. Horst J. Schirra
Cameron Anderson
Linda Kerr
Sheryl Maher
Jenny Rowland
Prof. Mike J. Waters
Prof. David J. Craik
Mater Children’s
Hospital
Prof. Francis Bowling
Teresa Munce
Dr. Gary Leong
Tony Huynh
School of Dentistry/IMB
Shaffinaz Abd Rahman
University of Ohio
Prof. John Kopchik
Queensland Smart
State Fellowship
Notas del editor
* Disorders of fatty acid oxidation and mitochondrial metabolism o E.g., medium chain acyl dehydrogenase deficiency * Disorders of porphyrin metabolism o E.g., acute intermittent porphyria (E80.2) * Disorders of purine or pyrimidine metabolism o E.g., Lesch-Nyhan syndrome (E79.1) * Disorders of steroid metabolism o E.g., congenital adrenal hyperplasia (E25.0) * Disorders of mitochondrial function o E.g., Kearns-Sayre syndrome (H49.8) * Disorders of peroxisomal function o E.g., Zellweger syndrome (Q87.8) * Lysosomal storage disorders o E.g., Gaucher's disease (E75.22)
Time courses of appearance of ethanol, acetate, formate - microbes or organic chemistry.
NST - non-sterile NMR tube, ST - sterile NMR tube
Jeremy Nicholson Nature Biotechnology metabonomic recommendations Beakering about the standards Heparin preferred for blood over citrate or EDTA as there is no interference with the analysis. Time of collection will affect the profiles and thus it is especially important with animal sample collection . Avoiding stress markers by acclimatizing animals to cages is essential Diet information of healthy controls should be as detailed as possible and fasted samples should be collected as for general testing Alcohol swabs should not be used prior to blood collection Things that have to be included in analysis part should include: design of experiment, data scaling, data normalization, algorithm selection, unsupervised statistical methods, supervised models, models testing and model validation