week 5 part 1 Flashcards
Define metabolomics
systemic identification and quantification of the small molecule metabolic products (the metabolome of a biological system(cell, tissue, organ, biological fluid or organism) at a specific point in time
What are metabolites?
small (<1000 atomic mass unit (amu x10-24g) chemical compounds (<1000 atomic mass unit
Define metabolism
Ensemble of chemical transformation carried out in the living tissue
What is netabolomics essentially extension of?
proteomics to the activities of expressed enzymes
Define metabonomics
subset of metabolomics involving the quantitative measurement of the multi-parametric metabolic responses of living systems to pathophysiological stimuli or genetic modifications
What are metabolites seen to be?
Most revealing markers of disease or chronic expsoure to toxins from the environment and of the effect of drugs
What are metabonomics a subset of ?
Investigation of the absorption, distribution, metabolism and excretion (ADME) OF DRUGS
What does metabonomics include?
not only intracellular molecules but also the components of extracellular biofluids
What does metabonomics deal with?
A diverse set of metabolites even more varied than proteins
Why is metabolomics not a new idea?
- Ancient physicians tasted patient’s urine for sweetness to test for diabetes
- Urine charts were for diagnosis in middle ages
- Blood lactate is a diagnostic and prognostic tool for newborns with perinatal asphyxia
What is used to measure the degree of lactic acidosis?
- lactate and not base deficit
What did the study examine?
The relation between blood gas measurements and outcome following perinatal asphyxia (pre-term infants)
What can urine lactate be?
Novel biomarker of lactate production capacity but reliability has been unsatisfactory so far
What can metabolomics provide?
- unbiased approach to assess a process without prior assumption
What are metabolites?
- end products of all processes occurring in cells, including gene, transcription and protein function
- provide a snapshot of biological status
what causes substantial changes in metabolites levels?
- subtle changes in gene, transcripts and protein
What is used to analyse blood metabolites?
NMR spectroscopy
What is example of endogenous metabolite?
normal cellular function
What is example of Exogenous metabolites?
- Diet
2. Gut flora
how many known metabolites are there?
- 200,000
How are compounds identified?
comparison with known standards within library (National institute of standards and Technology)
How long does it take a laboratory to identify one metabolite
A day by looking at 500 spectra per day
What can recent algorithms do?
Sift through 1000x times more
What is mass spectrometer?
Instrument which can measure the masses and relative concentrations of atoms and molecules
What is GC-MS?
- separate compounds using gas chromatography
- Detection using mass spectrometry
- Separation by GC based on volatility and polarity
What is LC-MS?
- separate compounds using liquid chromatography
- Detection using mass spectrometry
- can use higher molecular weight compounds than GC
What is LC-MS/MS?
- liquid chromatography tandem-mass spectrometry
- the first MS analyses the precursor ion
- The precursor is fragmented and is analysed with a 2nd MS
What is advantage of MS/MS?
- increases sensitivity
2. more structural information of the analytes
What does LC/MS provide?
- further selectivity
- unbiased detection
- information about the structures of the separated compounds
How are metabolites introduced into MS?
- electron spray ionisation (ESI)
2. enter as charged molecules that are transported in an electrical field between end of columns and entrance of MS
How can ionisation occur?
- Deprotonation (ESI-)
- Protonation (ESI+)
- Results in single or multiple ions
- structural information is obtained by collision induced decomposition
What is mass spectrometry?
- intrinsically a highly sensitive method
2. powerful tool for detection, quantification and structure elucidation of metabolites
What is the most popular approach for metabolomics application?
- LC-MS
What does GC-MS achieve?
a better metabolite seperation and generally avoids ion suppression
What are steps to mass spectrometer?
- Ionisation
- Acceleration
- Deflection
- Detection
What is Ionisation?
- the atom is ionised by knocking one or more electrons off to give a positive ion
- mass spectrometers always works with positive ions
What is acceleration?
- the ions are accelerated so that they all have the same kinetic energy
What is deflection?
- The ions are then deflected by a magnetic field according to their masses
- the lighter they are, the more they are deflected
What is Detection?
- The beam of ions passing though the machine is detected electrically
What is NMR?
- less sensitive than MS
- Highly quantitative
- quantify metabolites in intact tissue and tissue extracts
What are the characteristics of NMR?
- rapid
- non-destructive
- Requires little sample preparation
What is NMR good for?
Determining structure of unknown compounds
What can certain nuclei(e.g. Proton (1H)) give?
distinct signal if excited by specific radio frequencies
What are the typical approaches?
- use chromatography to separate components of interest
2. use mass spectrometry to identify them
What does the peaks in NMR spectra show?
- reliably assigned to specific metabolic species
2. provides a wealth of information on identity and quantity of large number of metabolites
What are NMR-based metabolomics approached used to study?
- effect of:
- drugs
- toxins
- various diseases - to trace metabolic pathways and measure fluxes
What does non-invasive nature of NMR provide?
unique opportunities to translate in vitro findings to clinical applications in vivo
What is it impossible to look at?
- whole metabolome
2. look at 1 type of metabolite
What are examples of diverse chemical compounds?
- ionic inorganic species
- Hydrophilic carbohydrates
- Volatile alcohols
- Ketones
- Amino and non-amino organic acids
- Hydrophilic lipids
- complex natural products
Why is it difficult to study metabolites?
- Typical half-life is often in seconds or less
2. require rapid quenching of all biochemical processes for all samples
What is Global metabolic profiling?
- untargeted metabolomics
- profile all metabolites in the sample (measuring hundreds to a few thousand metabolites in a single analytical run) which includes both known and many unknown metabolite peaks
Why is global metabolic profiling a good approach?
- involve discovery research
2. provide a starting point to identify unique metabolic changes or generating biological hypotheses
What is Targeted metabolic profiling?
- A set of known metabolites are quantified
- Hypothesis driven approach in which pre-defined metabolites are chased based on available databases - use standard compounds
- resulting data used for pathway analysis to prove or disprove biological hypotheses/Variables for statistical analysis
What does metabolomics generate?
- information for small molecule metabolites in a global snapshot of studied metabolism
What does univariate analysis include?
- P values
- Welch’s T-test
- useful to identify individual biomarker
What are examples of multivariate analysis?
- principal component analysis (PCA)
2. Partial least squares (PLS)
What does MVA enable?
Identification of the most variable/separation and put forward for further analysis
What is Parkinson’s disease?
- age-related neurodegenerative disease
2. positive response to L-dopa treatment
What are the symptoms of Parkinson’s disease?
- Tremor
- Rigidity
- Bradykinesia
- speech issues
What has few studies used metabolomics to evaluate?
metabolic signatures and pathways invplved in parkinson’s disease
What has been shown to be related to Parkinson’s disease with Parkin KO mice?
The energy metabolism pathwya
What was the first causal link to Parkinson’s disease?
A53T (Alanine-Threonine) missense mutation
What did overexpression of human A53T mutation in mice cause?
- Nigrostriatal neuronal loss
2. Alpha-synuclein aggregation
what has been affected the most in post-mortem brains of PD patients?
- The pentose phosphate pathway
What is significantly increased in plasma of PD patients
Glutathione
What was collected for metabolomics profiling?
- A53T transgenic mice
2. Age-matched non-transgenic (nTg) controls
What was all mouse work approved by?
Institutional Animal care and Use Committees
why was the forebrain and midbrain tissues collected?
Midbrain is where the substantia nigra is located
What did samples undergo?
- aqeueous methanol extraction to remove the protein fraction to extract the small molecules
What did the extracted sampled have removed?
- solvent
2. Frozen and then dried under vacuum ready for global untargeted metabolic profiling
what was used to quantitatively compare the global metabolomics profiling data collected from the 4 groups of mice?
- principal component analysis (PCA)
What did PC1 and PC2 account for of overall variability?
- PC1 - 27%
2. PC2 - 13%
What is FDR?
- A commonly used filtering criterion for large-scale data analysis
what did the test allow us to capture?
maximum number of compounds that potentially changed between groups
58 metabolites were significantly disturbed during aging
in the aged vs young mice what metabolites were increased or decreased?
- 25 metabolites were increased
2. 33 metabolites were decreased
What separated the age groups?
- Hierarchical clustering of the 58 metabolites
What was altered which reflected the energy demand in the mouse brain?
- Alanine metabolism pathway
What was used to identify which pathways were altered by the 58 metabolites?
- metabolite set enrichment analysis (MSEA)
What was the best pathways to predict age affect in?
- Redox homeostasis
- Lipid synthesis
- Amino acid metabolism
What biosynthesis pathways were disturbed in the aged mouse brains?
- beta-alanine metabolism
- glutathione metabolism
- pantothenate and COA biosynthesis
what is affected by aging only?
- Alanine
2. acetyl COA
What is affected by aging and A53T mutation?
- Guanosine metabolism
What did further analsysis reveal?
guanosine accumulated more in the aged A53T mice compared to young ones
- no substantial alterations of the guanosine level found in young and aged nTg subgroup
What was guanosine affected by?
- combination of genotype and age factors
2. confirmed by two-way ANOVA interaction test
What can Guanosine be phosphorylated into?
- GMP
- GDP
- GTP
Involved in cellular growth, differentiation and survival
What does guanosine have?
protective effect against stuarosporine or beta-amyloid-induced apoptosis
What does Guanosine have neuroprotective effect against?
- MPTP
2. 6-OHDA induced apoptosis
What pathway is involved in aging?
- Alanine metabolism
2. Acetyl-cOA biosynthesis
What has been considered as a biomarker for PD diagnosis>
- Uric acid
2. the end product of purine pathway
What are the several techniques available to study metabolite profiling?
- High performance liquid chromatography (HPLC)
- Gas chromatography (GC)
- Optical spectroscopic (OS)
- Nuclear magnetic resonance spectroscopy (NMR)
What is NMR spectroscopy easy?
- rapid metabolite detection
- involves uncomplicated preprocessing procedure
- reproducible and quantitative
What was carried out on normal and drug-naive parkinson patient to identify
- 1H(proton) NMR spectroscopy to identify 22 targeted metabolites in blood plasma
What was the method for this study?
- 43 patients
- 37 age and sex-matched controls were recruited from India
- Blood samples (4ml) were collected in EDTA vacutainer tubers
- plasma was collected and stoed at -20 until used
- 500 microlitre plasma was processed by Nanosep 3KD microtubes to extract the metabolites from samples and avoid protein interference
- metabolites were dissolved in heavy water (D20) containing hydrogen isotope deuterium and internal standard
What was samples subjected to
- 1H NMR spectrum
What was 22 targeted metabolites in plasma analysed using?
Chenomx software
compared to library of 292 metabolites
what software was used to carry out ANOVA?
Genespring
What software was used to carry out partial least square discriminant analysis
umatrices software
What was used to identify interacting genes?
pubgene index
What was significantly decreased in Parkinson’s patients vs control?
- Galactitol
- Glycerol
- Methylamine
- Trimethylamine
- Ethanolamine
- Suberate
- Glutarate
- Malate
- methylmalonate
- succinate
- acetate
- gluconate
- threonate
14, glucolate
- ascorbate
- isocitrate
What was increased in Parkinson’s patients vs control?
- ethymalonate
- pyruvate
- myoinositol
- sorbitol
- propylene glcyol
What did heat map show?
The average difference in concentration of metabolites between normal and patients
What metabolites was increasd in parkinson’s patients vs control
- Myoinositol
- Sorbitol
- Pyruvate
What was decreased?
- Citrate
- Acetate
- succinate
What is increase myoinositol level related to ?
decreased motor nerve conduction velocity and neurological diseasse
what is increase in sorbitol level related to?
impairment in oxidative stress
What are Myoinositol and sorbitol involved in?
polyols metabolic pathway that can be altered in mitochondrial dysfunction
What is the end metabolite of glycolysis?
pyruvate
What are intermediates of Kreb’s cycle and what does it demonstrate?
- Citrate
- Malate
- Succinate
changes in pyruvate dehydorgenase activity
