Module 7 - Investigation of Metabolism PDF

HSS 4324 - Research Approaches in Health Biosciences Yan Burelle, Ph.D. Professor Interdisciplinary School of Health Sciences, Faculty of Health Sciences & Department of Cellular and Molecular Medicine, Faculty of Medicine University Research Chair in Integrative Mitochondrial Biology University of...

HSS 4324 - Research Approaches in Health Biosciences Yan Burelle, Ph.D. Professor Interdisciplinary School of Health Sciences, Faculty of Health Sciences & Department of Cellular and Molecular Medicine, Faculty of Medicine University Research Chair in Integrative Mitochondrial Biology University of Ottawa Pavillon Roger Guindon Room 2117 451 Smyth Road, Ottawa, Ontario K1N 8M5 Lab website : www.burellelab.com Phone (office) : 613-562-5800 ext 8130 Investigation of metabolism • Understand the main methods to study whole body metabolism (direct and indirect calorimetry) In vivo method to look at metabolic rates and engird expenditure more focused techniques • Understand key methods to study metabolism at the organ level and cellular level: metabolic imaging, isotopic tracing, metabolomics, tissue biopsy, ex vivo organ perfusion Various levels of biological complexities • Understand basic methods to study mitochondrial metabolism and bioenergetics isolated mitochondria, permibilized cells, etc. Investigation of metabolism • Metabolism is all about energy transfer Nutrients (ex: C6H12O6) O2 Glucose - carbohydrate injected from meal combusted in presence of oxygen Extract the high energy contains in the carbon hydrogen bonds high energy shuttle molecules generated to fuel mitochondrial respiratory chain (H+ Reduced equivalents (Redox Energy) and electrons from nutrients are recovered as NADH et FADH2) Everytime there is an energy exchange, second law of thermodynamics applies; energy exchange is not 100% efficient and a certain proportion is lost as heat • this is why our body temp is 37 degrees • efficiency of body in transferring redox energy to phosphate is 60-70% so 30% loss of energy as heat Heat Redox energy - Converted into phosphate energy that is useful on our cells (ADP —> ATP) ATP (phosphate energy) Heat Work (mecanical or other) Synthesizing DNA, pumping ions, etc. Investigation of metabolism Nutrients Amino acids Carbohydrates Glycolysis Enter various metabolic pathways at different levels ATP Pyruvate Acetyl-CoA CAC NADH2 – FADH2 (reduced equivalents) to all types of nutrients fuels rest chain to make ATP in presence of oxygen NH3 CO2 Mitochondria Generates acetyl-CoA and FADH and NADH GTP ATP Oxidative phosphorylation • Some metabolic pathways are however common: • Krebs cycle • Oxidative phsophorylation Fatty acids b-oxidation • Nutrient catabolism occurs through distinct metabolic pathways • Glucose = glycolysis • Amino acids = transaminations and deaminations • Fatty acids: b-oxidation O2 H2 O Investigation of metabolism Whole body Have many techniques depending on the level of investigation Organs Cells Intracellular organelles Investigation of metabolism Direct calorimetry Indirect Respiratory calorimetry Measure heat production Whole body Organs In vivo: metabolic imaging, isotopic tracing, metabolomics, tissue biopsy Ex vivo: organ perfusion Cells Extracellular flux analysis, metabolic tracing, metabolomics Permeabilized cells, isolated mitochondria, respirometry Intracellular organelles Direct calorimetry • Combustion of a fixed amount of nutrient allows to determine its energy content energetic value • This is how we know the caloric value of foods (4, 4, et 9 kcal/g for sugars, proteins and fat respectively) Bomb calorimeter 100 % heart Efficienmcy= 0 % insluated box, w/ water, thermometer, and magnetic stirrer to mix up water, and combustion chamber in water bath with small place to put food • ignition device has oxygen and lights up molecule and it burns, and frees energy contained in nutrient • 100% will be released as heat and increase in water temperature is noted 4 kcal 9 kcal 4 kcal Oxygen 1 g glucose 1 g palmitic acid (lipid) 1 g protein (amino acids) Direct calorimetry: The caloric bomb for humans Direct calorimeter to look at whole body metabolism • Subjects are put in a insulated closed-circuit room • Heat production is measured directly Allows input of constant supply Very sensitive device to measure small changes in temperature • Requires a complex and expensive facility • Can be combined to an O2 measuring device insulated room: air inlet and outlet w/ filter to absorb CO2 and O2 and recycle air • walls of these chambers are filled with pipe in which water is circulated with two chemo,eteres (energy and exit); as water circulates in the walls, it will be heated by heat produced by ppl inside • by measuring difference in temperature in inlet vs. outlet, you can determine how much heat the guy in the caliometer has produced Indirect calorimetry more frequently used Measure O2 and CO2 exchange to calculate energy expenditure • Mask or mouthpiece is used to collect expired gases • O2 and CO2 concentration in inhaled and exhaled air is measured by mass spectrometry so we can have precise values on how much oxygen and CO2 are being consumed and relieved • Ventilation rates (in L/min) are measured using a flowmeter In the mask or outlet Monitor energy expenditure at rest • This allows to calculate VO2 i.e. the volume of oxygen consumed per min and VCO2 i.e. the volume of Carbon dioxide produced per min • From these values it is possible to calculate rate of carbohydrate and fat oxidation (proteins neglected) in the body and energy expenditure two most important energy substrates that we use to produce our energy on a daily basis • proteins often neglected as they are a minor source (no more than 15% of energy expenditure is from proteins) Indirect calorimetry Calculate how much of each substrate is being oxidized Glucose C6H12O6 + 180 g/mol 6O2 6CO2 + 22,4 L/mol Palmitic acid (lipid) C16H32O2 22.4L/mol CO2 133.4 L/mol glucose + MW: 256 g/mol 23O2 6H2O – 686 kcal/mol glucose Glucose’s Potential Energy= 3,8 kcal/g e.g. ~ 4 kcal/g O2 energetic equivalent = 5,10 kcal/L How much energy to I release when I consume 1 L of oxygen VCO2/VO2 (i.e. respiratory quotient RQ)= 1 equal in the case of glucose 16CO2 + 16H2O – 2398 kcal/mol Palmitate’s Potential Energy = 9,36 kcal/g e.g. ~ 9 kcal/g O2 energetic equivalent = 4,65 kcal/L VCO2/VO2 (i.e. respiratory quotient RQ)= 0.70 Require far more oxygen than you release CO2 These equations tell us: Burned • The exact amount of O2 consumed and CO2 produced when each of these nutrients are oxidized • The exact amount of energy produced from the oxidation of these nutrients Only measuring VCO2/VO2; can back calculate everything else; ex. glucose oxidized, energy produced, etc. Contains far more energy since it is a 16 carbon molecule Indirect calorimetry In daily life, we use a mix of substrate (fats and carbohydrate) to produce our energy • rare occasions where we only use glucose (ex. Intensive exercise) • If VCO2 and VO2 (and thus the RQ) are measured, the table of respiratory quotients can be used to establish the contribution of fat and carbohydrate to energy production • The O2 energy equivalent (EqO2) depends on the mixture of substrate used by the body and ranges between 4.851 and 5.2 kcal/l of O2 comsumed. • Can measure rate of oxidation of aft and glucose and how much energy you are deriving from each one If you are using mix of fat and carbs to produce energy, RQ will be somewhere between min (0.70 for fat) and max (1.0 for glucose) % E glucose 0 3 7 10 14 17 21 24 28 31 34 38 41 45 48 51 54 58 61 64 67 71 74 77 80 83 87 90 93 96 100 % E fa 100 97 93 90 86 83 79 76 72 69 66 62 59 55 52 49 46 42 39 36 33 29 26 23 20 17 13 10 7 4 0 RQ 0.70 0.71 0.72 0.73 0.74 0.75 0.76 0.77 0.78 0.79 0.80 0.81 0.82 0.83 0.84 0.85 0.86 0.87 0.88 0.89 0.90 0.91 0.92 0.93 0.94 0.95 0.96 0.97 0.98 0.99 1.00 kcal/L O2 4.851 4.861 4.874 4.884 4.897 4.907 4.920 4.930 4.944 4.954 4.964 4.978 4.988 5.002 5.012 5.023 5.033 5.047 5.058 5.069 5.079 5.094 5.104 5.115 5.126 5.137 5.152 5.163 5.174 5.185 5.200 kJ/L O2 20.37 20.41 20.47 20.51 20.57 20.61 20.66 20.71 20.76 20.81 20.85 20.91 20.95 21.01 21.05 21.10 21.14 21.20 21.24 21.29 21.33 21.39 21.44 21.48 21.53 21.58 21.64 21.68 21.73 21.78 21.84 Indirect calorimetry Oscillation of RQ Devices measure VCO2 and VO2 and calculate energy expenditure in kill per hour Mice are nocturnal Sleeping Day time Low activity - energy expenditure is low Increased reliance on fat, which doesn’t release energy as fast as glucose • RQ is 0.85 meaning mice are using a 50% mixture of fat and glucose during the day Can do similar experiments in homes and measure VO2 Max (Ex. If you’re an althelete and want to know your maximal capacity) • basis for calculating things related to nutrition, exercise performance, dietary plans, etc. Active at night High activity of metabolic rates Increased reliance on Carbs faster way to produce energy • RQ is 1.0 meaning mice are serving most energy from burning only glucose • • Can calculate energy expenditure form RQ table Can compare energy expenditure between control and experimental Indirect calorimetry Indirect respiratory calorimetry: summary Nutrient oxidation equations • VO2, VCO2, urea ----> glucose, fatty acids and protein oxidation rate ---> energy produced/consumed For protein oxidation based on urea excretion • Track energy expenditure dynamically changes over minutes or between breathing cycles (rest to excersize to rest again) • Track usage of various energy fuels • Non invasive, easy to use, technique applicable in flies and elephants. • Limited information on metabolism of specific tissues. We do not know how much the brain is consuming, the liver is consuming, etc. • No information on metabolic pathways (black box model: we only measure O2 consumed and CO2 produced and infer the rest) Good for global view but limited when it comes to examining organ metabolism Organ metabolism Old school methods Oxygen level in artery is higher in than in vein • difference is related to amount of oxygen that the muscle consumed • if you know diff in concentration of oxygen between artery and vein and the blood flow, you can calculate the VO2 of quad muscle (Ex.) • can know amount of CO2 released, and amount of glucose consumed • A-V difference (O2, CO2, circulating substrates (e.g. glucose)) Combined to: • Tissue biopsy (metabolite level measurement preDo this repeatedly over time, and take glycogen content, you can measure of glycogen consumed by muscle in a certain excersize by post) amount measuring initial and final amount • Invasive • Not applicable to all organs Easily applicable to muscles • can apply to liver but it is pretty invasive Ex. Brain metabolism • no biopsy is possible in brain Exercise physiology lab • measuring muscle metabolism of quad muscle • guy is doing leg extension and has catheter and biopsy being taken • catheter in femoral vein (look at what’s coming out of the muscle) and artery (input) • Arterial venous difference: artery - what’s left in the vein and used to see what has been consumed or released by model • can measure blood flow Organ metabolism Isotopic dilution techniques Example: Circulating glucose turnover measured using stable isotopes Can track dynamics of how certain nutrients are being used by using tracers Trace amounts of labeled glucose in injected at constant rate through IV infusion pump • Blood samples are taken at regular intervals through another IV line *Ra glucose • Relatively non-invasive • Allows to quantify liver and peripheral tissue glucose metabolism 2H 2O Glucose used by other organs (Rd) • oxidized to CO2 or stored as glycogen constant infusion known isotopic enrichment R = Rate of appearance, Rd = Rate of disappearance infusion Glucose (mM) When stable enrichment is achieved the level of enrichment is proportional to the rate of appearance and disappearance of endogenous unlabeled glucose [6,6 2H]-glucose *Rd glucose Blood glucose is highly maintained (5 millimil per litre of blood) • “disappearing” glucose is replaced by glucose being put in circulation by liver (Ra) • flux of glucose form liver to perhiphirla organs transmitted through the bloodstream (i.e 2H/H ratio) Put catheter in veins and inject small amount of labelled glucose (Rd) (Ra) • once equilibrium is reached, can use rate of infusion to derive Ra a and Rd of glucose • ex. If we start performing At this point, any changes in the 2H/H ratio will be due to changes in Ra (i.e the source of unlabelled glucose) excersizwe, liver will produce more glucose as body uses up more to maintain glycemic, Only relies on blood sample and isotopic enrichment will drop and reach new steady state: progressively precisely more orange beads • can use to evaluate if a person is diabetic/ than yellow beads not, etc. since you are infusing at constant rates • can be detected by mass spectrometry Time Isotopic enrichment • unlabeled glucose CO2 infusion equilibrium will be reached if person is sitting/at rest Time Organ metabolism Metabolic imaging Label injection (molecule bearing a stable isotope i.e. 2H or 13C) Way to get regional vacation in the same organ about how metabolism differs in vearous areas useful in context stroke and Brian injury or neurodegenerative diseases to study altered brain metabolism Deuterium metabolic imaging (DMI) of brain glucose metabolism in vivo Two labels injected [6,6ʹ-2H2]glucose or [2H3]acetate Magnetic resonance imaging and spectroscopy Glx = Glutamate Small animal MRI Imaging of brain in a mouse In each box, measure concentration of glucose, glutamine, or lactate in various regions of brain Human MRI • gives spatial information on tissue of interest combine imaging to mass spectrometry Anatomical image To get a view of the organ in vivo + Metabolic intensity map To detect metabolites of interest Can detect water, glucose, glutamine, lactate, etc. Each box is a volume of the brain that reflects metabolism in that part of the brain • if you sum them up, you have the spatial map of glucose metabolism for example. Peak maps that reflect intensity of metabolism of specific molecule - more intense - red, less intense = blue Image G: lactate metabolism very intense in this part of the brain - this region is probably performing a lot of anaerobic glycolysis and aerobic glucose metabolism is shut down De Feyter et al.Science Advances 22 Aug 2018: Vol. 4, no. 8, eaat7314, DOI: 10.1126/sciadv.aat73 Organ metabolism Ex vivo organ perfusion perfused mouse heart • Organ metabolism can be studied ex vivo using perfusion systems Only used in preclinical studies, in animals and in extracted human hearts post translation • Conditions can be strictly controlled • Functions can be measured In the heart: pressure produced, cardiac output Perfusion rickets going to be done • performs work an you cantata samples of the bugger in and out and measure oxygen consumption, glucose metabolism, etc., Luver function: rate of glucose production, synthesis of glycoproteins, HDL, etc. • Metabolism can be analyzed using a variety of techniques • Basic biochemical assays • Isotopic tracers • Metabolite analysis by mass spectrometry (i.e. metabolomics) To look at glucose or other metabolites • Molecular tools can also be used to look at genes and proteins Can sample tissue at end of experiment and process it to do so and couple information together • However: Portal vein, hepatic vein, etc. are cannulated, and you can measure blood flow through the device; can push blood at certain rate in liver and do AV difference and look at glucose production, fatty acid consumption, etc. • Invasive and thus mostly restricted to pre-clinical animal models • Partial or total loss of in vivo regulatory complexity Organs are no longer innervated, don’t receive hormones, etc; limited in mimicking real life situation but provides advantages Isolated mouse liver perfusion Cellular metabolism Cellular respiration • Measuring respiration is a great way to examine metabolic perturbations within cells. measure respiration in a cardiac cell from diabetic vs. Healthy patient • there are differences • A variety of test conditions can be devised to pinpoint which metabolic pathway(s) and parameter(s) is altered in a given situation ex. In diabetes, it could be. Genetic mitochondrial disease that affects the respiratory chain • The downside is that tests are performed in an in vitro setting, which poorly mimics some in vivo conditions. In vivo In vitro Cardiac muscle cell - do histology on it, you can see that cardiomyoctes are attached to each other thru intercalated discs; they’re all sewn together, tightly packed, and assembled in an extracellular matrix • tissue works as synthesis meaning they all contract simuntanoulsy, etc. • capillaries inside to circulate blood around nerve endings that provide signals for cell to contract If you treat heart tissue w/ enzymes to dissociate the cells, you don’t get individual cardiomyocyctes You can see actin and myosin filaments, sarcomeres, are all dissociated; intercalated discs are dissolved and they stand alone as single cells • but they are floating around alone which is far from reality Cellular metabolism Standard Oxygraph Cell suspension not permeable to fluid - easily detects changes in oxygen concentration inside the cell suspension Inserted inside chamber, then we close lid Monitors oxygen content in the chamber small capillary through cap where you can inject stuff Cap Oxygen-permeable membrane Capillary Allows electrical conduction between the plus and - side of the electrode KCl electrolyte Anode Cathode Heater Double walls on chamber to control the temperature - insulated and there is a heater important to keep cells at 37 degrees for ex. Cell suspension sample Chamber magnetic stirrer inside to keep the cells homogeneous/ floating around nicely in chamber Oxygen sensitive electrode Records oxygen concentration within the cell suspension Cell Electronic board w/ magnetic stirrer and motor Cellular metabolism Standard Oxygraph • Graph show O2 content in the test chamber (in nmol/ml) In lid, small hole where you can insert a needle or pipette and inject stuff • Negative slope indicate that cells consume the O2 available. Standard oxygraphy works well but: • Has a low throughput (i.e. 1-2 test at a time) • Takes a lot of cells (~5 M per test) • Only work with cells in suspension not suitable for every cell, ex. primary neurons (don’t like to be non-adherent) Cells are added Once there is a stable baseline • close lid At the beginning: oxygen content is stable in the chamber - nothing in the chamaber that consumes oxygen was added yet • lid is open Negative slope • proportional to rate that cells added in chamber consume oxygen • by measuring slope of this curve, you can determine rate of oxygen consumption x-axis: time Cellular metabolism Extracellular flux analysis Can be used in clinical setting or more basic research volume of chamber is very small - don’t need a lot of cells to pick up a single • Allow for the analysis of respiration in a small number of adherent cells in multiple wells (up to 96) simultaneously only need 25,000-100,000 cells per well instead of several millions At bottom of culture dish Oxygen sensing system is much more sensitive Size of chamber: 10 micrometer thousandfold smaller than oxygraph Cell culture plate - conical culture well Put cells in cell culture incubator w/ this plate • on day of test, take them off incubator and put lid on top • each well has detector on cap; there will be a probe that will come in and send signals PO2 (mm Hg) micro chamber forms if probe is lowered and ring is formed • pick up changes in O2 concentration in sealed microchamber 10 µL cells cultured adherent in micro chamber measure 10 wait Probe is lowered again mix Probe is lifted here before oxygen content is completely depleted 5 0 cells 25-100k down up 15 Can put in drug to inject in your cell culture experiment chamber sealed when probe is lowered • oxygen concentration will decrease during that period as there is no oxygen admission • probe will detect drop in oxygen content in microchamber • decreases rapidly since chamber is very small so Can perform several cycles of up and down and there is only a average the slope to get rate of oxygen consumption tiny amount of stable state oxygen there 0 500 Time (sec) 1000 Cellular metabolism Cellular Oxygraphy • Measuring basal oxygen consumption of cultured cells gives an idea of oxygen requirements per cell. However, it does not tell you: • • • • • Is it solely mitochondria or are there some oxidases responsible for consuming the oxygen Where is the oxygen being used? Is it working at maximum? Don’t know if it is breathing at 10% of its max or 100%, etc. In cell culture media, there is glucose, What substrates are being used? lipids, amino acids, etc. What is limiting oxygen consumption? Is it at the level of mitochondria or elsewhere? How efficiently is it being used in terms of ATP production • Some of this information can be gleaned from adding specific substrates, metabolic inhibitors and uncouplers. Rate of glycolysis assessed by change in pH Rate of respiration assessed by changes in oxygen concentration Cellular metabolism Cellular Oxygraphy • Basal respiration Blocks complex 5 and shuts down ATP synthase • difference between this rate and basal respiration is the rate of O2 Once measurement is steady, inject consumption that was devoted another drug to the synthesis of ATP • Oligomycin – inhibits ATP synthase • To gain further knowledge, use injection ports to sequentially inject different inhibitors O2 consumption due to ATP synthesis and use 3-4 fold above basal blows holes through the membrane - drives respirator chain; pumps protons to try and reestablish electrochemical gradient, but there is a hole in the membrane so the respiratory chain will go crazy • FCCP – uncouples oxygen consumption from ATP synthesis • Measured 3 times under same condition to make sure we have reliable measurement Maximal O2 consumption capacity Illicit maximal capacity of respiration of these cells Difference between spontaneous rate of respiration and the maximum they can reach Reserve capacity expressed in fold over basal Entirely • Antimycin-A & Rotenone – inhibit respiratory chain • Residual O2 consumption not related to mitochondria • Proton leak: mitochondrial respiration unrelated to ATP synthesis protons accidentally make it through the membrane as membranes are leaky - protons reenter without making ATP • don’t know what oxygen is being used for at this rate ex. Ppl w/ mitochondrial disease have maximal respiration close to basal even at rest Mitochondrial function If there is a problem with transport of these inside cell, mitochondria might respire less • In order to specifically investigate mitochondria, full access to these organelles is required: Confounding factor: Intact cells are not the best to look at mitochondria because there might be some events in cytoplasm or plasma membrane that indirectly ends up affecting oxygen consumption rate Control over substrates Control over ATP/ADP • This allow much better control on experimental conditions • Use specific mitochondrial respiratory substrates that cannot enter intact cells • Control on local concentrations of substrates Ex. NADH or kerb cycle intermediates that normally feed directly the respiratory chain ATP Endogenous substrates Permeabilize with mild Makes holes in cellular detergent membrane • We can permeabilize plasma membrane to access them • Or use centrifugation to prepare isolated mitochondria ADP Since there is a lot of holes, cytosol content comes out and you can start adding substrates that are specific to their respiratory chain or isolate by centrifugation Mitochondrial function Respirometry can help you to understand many aspects of mitochondrial respiratory function – tissue capacity for different substrates, electron transfer system dysfunction, degree of coupling… it is important to use the right protocol for your question one example: • Characterising electron transfer system (ETS) abnormalities Mitochondrial function Mitochondrial function Characterising electron transfer system (ETS) abnormalities Can dissect out where the respiratory chain is messed up with an oxygraph Block resp. again Bypasses Triggers ATP synthesis and complex 5 complex 1 will consume proton gradient and force and resp. resp. chain into action resumes bloicks resp. Chain Addition Role 1. Glutamate/malate Substrates for Complex I 2. ADP Activates OXPHOS 3. Rotenone Inhibits Complex I 4. Succinate Substrate for Complex II 5. Antimycin A Inhibits at Complex III 6. TMPD/Ascorbate Substrates for Complex IV 7. Cytochrome c Tests outer membrane integrity Specific substrates that deliver electrons Specific electron donors downstream of block Baseline resp. Can compare a control group w/ a patient group and if patient has isolated complex 1 defect, you can see that if respiration is lower than control w/ glutamate, but in succinate and TMPDS/Asc it becomes normal Kuznetsov et al (2008) Metabolomics Metabolomics vs other omics Genomics ~40 000 genes Genotype: Potential of the system Proteomics ~106 proteins Metabolomics ~20 000 metabolites enzymes and transporters involved in metabolic pathways Phenotype: Functional status of the system Reflects activity of metabolism Metabolomics is the omic science that is closest to linking genotype and phenotype Metabolomics Metabolomics vs other omics pathology stress circadian rhythms hormones mutations Metabolic levels affected by multiple factors; pathology, stress, circadian rhythm, nutrition, hormones, drugs, mutations, etc. Response Minute cxhnage sin the activities of cells • of you eat a meal, metabolites will change instanously but protein expression in genes might not • responsive to daily perturbations Response • Metabolomics therefore offers greater time resolution to detect short-term changes vs proteomics and genomics Response nutrition • The level of metabolites change more rapidly vs that of proteins and genes drugs Time Metabolomics Metabolome Some useful terms All metabolites Tissue extracts Body fluids Cellular extracts Metabolomics Blood, urine , any fluid you can extract from body Identification and quantification of metabolites in a biological system Biochemical pathway map Metabolites Organic compound that is either a metabolic intermediate or end product of restricted molecular weight (<1500 Da) Metabolomics Some useful terms • Standard metabolomics methods allow to determine the concentration of a metabolite. It is a snapshot image that does not inform on the flow (i.e. rate of synthesis and degradation). Not measuring rate of pathway fluxes • Fluxomics combines metabolomics methods with stable isotopes (13C etc…) to determine flux through specific metabolic pathways add this label to molecules we want to track Can pinpoint where metabolite is going and at what rate main flux is unidirectional from M1 to M4 Biochemical pathway has been diverted - much less production of M4 and glucose is being used for something else, for ex. Metabolomics Applications for metabolomics • Hold a a lot of promises in the field of personalized medicine And their impact on various biochemical pathways that are essential for life give us an open window on the global metabolic state of individuals Metabolomics approaches Number of metabolites 10000 Every single mass spectrometry single Non-targeted analysis: unidentified you are looking at entire metabolome - restricted metabolites, complex profileSince to basic research rather than clinical context Metabolomic need to do complex database searches to identify what metabolite might be 1000 Targeted analysis: Several classes of metabolites, complex profile ex. Lipid and glucose metabolism in diabetes - gives 100 Metabolomic fingerprinting more comprehensive fingerprint of the disease Targeted analysis: several metabolites of one class 10 Metabolomic profiling ex. metabolimics experiment looking at perturbations of glucose metabolism to study diabetes - quality of data could be a bit lwoer 1 Targeted analysis: one metabolite 0 500 1000 1500 Ex. Blood doping agency; respoible for running a test on blood samples correctly and screen for a restricted number of metabolites that are derived from steroid catabolism Quality of data (quantification, precision, sensitivity, identification) Closer to left side of x-axis: less precision - more relative abundance of metabolite when compared to another but detect a thousand metabolites vs. 1 (trade-off) Unique metabolite Metabolomics approaches Targeted approach Un-targeted approach • Limited # of metabolites (often known and pre-defined) • Large # of metabolites simultaneously • To answer a specific hypothesis or as a diagnostic test • For discovery. Hypothesis generating • Quantitative or semi-quantitative • Precise and reproducible ex. Blood doping test Observe in non-biased way, large # of metabolites • Exploratory method. Not quantitative relative changes in metabolite being examined • differential signatures • Analysis and data handling are complex More restricted to research Targeted metabolomics workflow Liquid chromatography device • separates metabolites according to their retention time in liquid chromatograph • then injected in mass spectrometer metabolites according to Data acquisition Seperate time they spent in a column 1 metabolite = 1 m/z and one retention time (RT) • GC or LS-MS or • NMR Or entire biopsies from healthy organs Relative abundance to extract biochemical intermediates Statistical analysis Various analyses depending on the objective: • Univariate/multivariate; supervised/unsupervised; correlation, covariance Output is a list of known metabolites: we know their identity and we calculate their concentration and perform statistical testing • are levels of metabolites different in control vs. experimental group m/z Data analysis Every metabolite comes out on a mass spectrometry as a peak with a distinct m to z ratio • each peak = single metabolite that has been operated prior to being injected in mass spectometer • height of peak refers to abundance of metabolite • compare peaks to database toindentify metabolites and their aubdnace • List of metabolites with defined RT and m/z M is time and Z is charge Short mention time: metabolites that come out first form the column Long retention time: metabolites that take a long time to come out from the column • Intensity for each metabolite (area This is the abundance of the metabolite under the curve of each spectra) + QC check • Calculation of concentration (ratio vs standard) relative abundance Untargeted metabolomics workflow Data acquisition • GC or LS-MS Gas or liquid chromatography or coupled to mass spectometer • NMR Metabolic interpretation: Pathway analysis Gene Ontology website Data analysis are not in the • List of unknown Some database metabolites each with a measured m/z ratio and a retention time Some signals, you know which metabolite they correspond to, and others are metabolic entities - a signal that you don’t know exactly what it corresponds to • include in analysis even if you don’t know what it is b/c when you compare your control vs. Experimental group, unknown metabolite might be different btwn them - in this case, you might devote time to identifying it and deriving biological explanation, as it is a very important new discovery Statistical analysis Principal Component Analysis (PCA) Volcano plot Downregulated metabolite Unregulated Y-axis: statistical significance X-axis: fold change Identify metabolites that differ between groups High sig: low p-value - tails of volcano plot Investigation of metabolism Direct calorimetry Indirect Respiratory calorimetry Whole body Organs In vivo: metabolic imaging, isotopic tracing, metabolomics, tissue biopsy Ex vivo: organ perfusion Cells Extracellular flux analysis, metabolic tracing, metabolomics Permeabilized cells, isolated mitochondria, respirometry Intracellular organelles