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Skip to 0 minutes and 10 secondsHi everyone, welcome to the end of the course video. We hope youve all enjoyed the course, it looks like you all have enjoyed the course from reading all the various comments and various questions its obvious that youve learned a lot as well, especially for those of you who havent come across metabolomics before, so its great to see so many of you interested in metabolomics and applying it potentially in your research as well.

Skip to 0 minutes and 32 secondsWeve had a number of really interesting questions which Im going to go through now, and weve picked some of those out just to go through because theyre some of the more common questions that weve had and theyve covered different areas across all four weeks of the course within here. So the first one, Could someone help clear up the distinction between two of the four classes of metabolites. This was talking about the difference between metabolites weve taken from external environment, and metabolites that we actually consume or synthesize within the body, for example.

Skip to 1 minute and 6 secondsSo, everything that happens within the body we tend to call that endogenous metabolism, so for example once glucose is in the body we obviously break down glucose to glycolysis pathways, so we would call that endogenous metabolism. But, for example, if we take a drug and we take that, we eat that, take it with a drink, once thats in the body and before its metabolised its still called an exogenous metabolite, because it hasnt been metabolised within there, but weve taken it in from the external environment, whereas exogenous metabolites and endogenous metabolites can be metabolised as well.

Skip to 1 minute and 42 secondsSo we generally classify metabolites that are only synthesised within the body as endogenous metabolites and then metabolites we take in from the environment as exogenous metabolites. Now, it starts to get a bit confusing if you think about food, most of the metabolites that we break down or synthesise within the body actually come from metabolites that were taken in from food, from drink, etc. as well. So there is a blurry line between what is endogenous and what is exogenous within there. So I hope thats answered the question, if not, ask it again and well come back to you.

Skip to 2 minutes and 19 secondsSomebody else was asking about the difference between untargeted, semi targeted and targeted studies, specifically they were looking at specific plant secondary metabolites. So untargeted metabolomics is very much if you dont know which metabolites are of interest, so were trying to measure as many metabolites as we can in a reproducible way and to identify those metabolites, and then to define which metabolites are statistically different between two classes, it could be somebody has a disease and a group of people that dont have the disease. The important thing there is that we do not know which metabolites are of interest.

Skip to 2 minutes and 53 secondsA targeted assay is very much when we do know which metabolites are of interest, so for example it could be the 10 metabolites present in a glycolysis pathway. Now those metabolites that are chosen can be part of the same pathway, or they can be very different metabolites. In 1 study weve performed in the Phenome Centre Birmingham, we had metabolites, we applied the targeted assay but we knew which metabolites were present, and they came from different parts of the metabolic network. So it isnt that necessarily for targeted assays they all have to be biologically or chemical structurally related, they can be very diverse chemical structures or different areas of metabolism or different metabolic pathways.

Skip to 3 minutes and 35 secondsSo, if you know which metabolites are of interest then the targeted, or potentially the semi targeted would be appropriate. Now the different between the semitargeted and targeted is, with the targeted you know which metabolites are of interest and youre only targeting those metabolites, whereas for the semi targeted method, the metabolites that youre targeting, especially with some of the kits that can be purchased for example from Biocrates, here you have a list of metabolites and you dont get to choose which metabolites youre analysing within there, thats already been chosen normally based on whether you can buy authentic chemical standards or not.

Skip to 4 minutes and 10 secondsSo untargeted is when you really dont know which metabolites are of interest, targeted is when you know exactly which metabolites are of interest and then semi targeted fits in the middle where if for example in this kits, if you know that amino acids are important then theyre present in those kits and you can use some of those kits as well. So the third question, from John Sharville, Can we have some examples of how this is affecting healthcare. So you gave an example there of troponin, and different measures of troponin and different biomarkers instead of using troponin within there.

Skip to 4 minutes and 47 secondsTroponin is a peptide so its not related to metabolomics per se, but there are examples of where metabolomics has been applied in the discovery phase, and has either now been translated through, so from that discovery phase into potentially the clinic, and theres examples of where were already applying metabolites. So for example glucose, well take a normal glucose tolerance test to measure insulin sensitivity or resistance, or potential for diabetes, its already applied and you can buy a nice, very easy to use small kit you can go to your local chemist and buy these for example, which just with a blood spot you place onto a strip and it measures your glucose levels and reports that back.

Skip to 5 minutes and 30 secondsSo thats really going from they found out that glucose is really important all the way through to clinical practice, or routine practice within there. Another example of that is inborn areas of metabolism, so the number of metabolites that have been assayed within the inborn areas of metabolism, so when youre born you typically will have a dry blood spot, or blood spot taken which is allowed to dry, and then the number of metabolites are assayed within there to look for inborn areas of metabolism. The panel, or the number of metabolites within that panel, is growing slowly as we go through, so it started off for example with acyl carnitines and organic acids, and its growing beyond that now as well.

Skip to 6 minutes and 11 secondsSo there are a number of examples within there, within the Phenome Centre Birmingham, we have one example, I cant give exact details on it, but we performed a discovery based study, we found about 40 metabolites that looked interesting, we then developed a targeted assay to validate those metabolites and looked to see how they stratify subjects out into different groups, and we validated about 15 of those metabolites, so 15 of those metabolites were still important in a very independent set of subjects within there, but we developed that targeted assay to do that. So discovery is only about finding those metabolites, and then you will move to more of a semi targeted or targeted approach within there.

Skip to 6 minutes and 50 secondsSo there are a whole range of different examples out there, theres a number of metabolites being named as biomarkers, but we have to test those and validate those really well before they get in to clinical practice. And the ones that are validated we dont necessarily actually hear about because theres IP and theyre patented, so until theyre actually out on the market we dont know how important they are. Margaret Bee, on the Phenome Centre Birmingham article has asked about the HUSERMET study. Specifically it talked about twelve hundred samples.

Skip to 7 minutes and 23 secondsSo, actually in the HUSERMET study we collected about four thousand samples from about four thousand subjects, so its one sample from each of those subjects across a whole range of age, gender, BMI, smoking status, etc., and the paper that we discussed in there is looking just at the first twelve hundred samples. Now weve collected data for all four thousand, but the paper thats published we just looked at the first twelve hundred, because we wanted to show the methods and how theyre applied etc. within there. There isnt any plans to look at all of the data, currently, but all of that data will be publicly available hopefully later this year if not early next year on the metabolites repository metabolites.

Skip to 8 minutes and 8 secondsOK, so David Stewart has asked, Would like a clearer explanation of how leucine and isoleucine are separated by column chromatography. So column chromatography is very much, if you think about liquid chromatography, so liquid chromatography you have liquid as the mobile phase, and then you have a solid stationary phase, and coated onto that solid stationary phase which is normally silica not always, but is normally silica youll have a stationary phase coated on to that, or youll use that bare silica, depending on the conditions that youre applying.

Skip to 8 minutes and 40 secondsSo if for example the best technique we can apply for separating out leucine and isoleucine is something called HILIC chromatography, and within that we have bare silica, and then we have an aqueous layer on top of that bare silica, and metabolites basically diffuse into that liquid layer which is absorbed onto the stationary phase. So we have an aqueous layer that isoleucine and leucine will absorb in and out of as it passes through the column. Now the time it takes for isoleucine and leucine to be absorbed and then released from that stationary phase, or from that aqueous phase within there will depend how long, or will influence how long it takes to pass through the column.

Skip to 9 minutes and 24 secondsSo because isoleucine, isoleucine is very much straight metabolite, and isoleucine is a branch metabolite, because of that the solubilities are slightly different in that aqueous phase, so isoleucine will be retained in that phase a bit longer than leucine. So leucine will be retained in that more than isoleucine, so this is why we can get this separation out over time. And actually, there are 6 other isomers, theres 6 other metabolites that have the same mass, and weve shown with HILIC chromatography that we can separate all 6 isomers with that.

Skip to 9 minutes and 54 secondsSo its very important to think about which actual, for example for liquid chromatography which chromatography stationary phase we apply, whether it should what we call the reverse phase, or whether it should be HILIC based, and actually the Phenome Centre Birmingham we apply both to all the samples that we analyse.

Skip to 10 minutes and 13 secondsOK, so David Stewart as well, thanks David for all your questions, was also asking about if you detect a biomarker by LCMS, how do we then take that through really to translation. So thats a really interesting topic. So, one thing you mention, would you have a liquid chromatography mass spectrometry system in a clinical chem lab just to measure this maybe one, two or five metabolites. Well metabolomics is very much focussed on the discovery phase, so looking at as many metabolites as we can and then analysing that data after weve collected it to find maybe theres one, two, three, four, five metabolites are the most important. After that point we wouldnt apply those untargeted approaches any more.

Skip to 10 minutes and 56 secondsWe would apply a more targeted approach, from that discovery study weve defined whats important, so we dont need to apply the discovery study anymore. So wed either develop a targeted method just to quantify those one to five metabolites, and then we would validate that as Ive discussed earlier in this video to the point of where youre getting to point of care, it might be that you take a sample and then that is transferred into clinical chemistry labs and analysed applying something called liquid chromatography triple quadrupole mass spectrometry, which is very good for quantifying metabolites at really low levels.

Skip to 11 minutes and 31 secondsOr, it could be a point of care test which is something like the glucose test that you can buy from, you know, a local Boots or local chemist, where its literally something that can sit, and you can collect a sample and analyse that sample right at the bedside.

Skip to 11 minutes and 47 secondsNow really, mass spectrometers are expensive, so we dont and we cant have hundreds of mass spectrometers next to for example the patients in the wards, so really we use the expensive equipment to identify whats important, and then we want to develop tests and validate the simplest tests that we can, the cheapest tests, so if were looking at more than 1 pound a sample then you have to start to wonder whether its an appropriate test, but that test should be easy, cheap, easy to use for anybody and easy for somebody to be trained in how to use that as well.

Skip to 12 minutes and 21 secondsSo, expensive for development and parts of validation, but actually when you get to the bedside you want something thats much more simple and easier to use.

Skip to 12 minutes and 34 secondsWeve been asked, a number of people have asked about statistical methods, so we talk generally about supervised and unsupervised methods, weve had some questions about which are the most commonly applied, is there a standard set of tools that we can use within there. Well, if you go out to different groups around the world that apply metabolomics, each of those has their favourite, and its not a favourite and its the only one that they use, but it very much depends also on the experimental design.

Skip to 13 minutes and 0 secondsSo, if for example if we think about just univariate statistical analysis, if you just have a population of people that have a disease and a population of people that dont, then univariate testing youd apply for that would be very different to if you have a smaller set of people but youre measuring changes over time.

Skip to 13 minutes and 19 secondsSo, there is no one single tool thats appropriate for all data analysis, and it very much depends on the biological question that youre asking, it depends on the experimental design, and it depends on the results that you want to get out of there, do you want to identify a small number of metabolites, or do you want to look at the biology of mechanisms, and the type of data analysis you would apply for that can be different as well.

Skip to 13 minutes and 42 secondsSo theres no 1 standard technique, we talked about the common techniques that are applied for univariate and both supervised and unsupervised multivariate, but there are methods as well, other methods available, for example random forests, Support Vector Machines, SVMs, a whole range of other techniques as well which are used by, the more advanced processes, which are used by groups that can apply those.

Skip to 14 minutes and 10 secondsSo finally, also from David Stewart, thank you very much David youve talked about the identification of metabolites, it seems to be one of the biggest problems, and can the team talk about any new lateral thinking approaches to this. So when metabolomics started, about 20 years ago, we didnt have the technologies and we didnt have the information to know if we take a blood sample or a urine sample, the total parts list of each of those samples, and were slowly building that up, so theres databases like the Human Metabolome Database, with subsets so that you can define whats present in urine serum for example. So we are slowly building that up.

Skip to 14 minutes and 47 secondsNow the problem is that nobody runs the same methods, so a group here, we will apply an analytical method thats different to a group a hundred miles away in Manchester, for example, so standardisation is need and then once we have standardisation basically we can collect the analytical standards, the chemical standards that we need, we can define what the retention time is, we can define what the MS/MS mass spectrum is, it can be reproducible, we can show that its reproducible and therefore methods can be transferred across different laboratories, and this metabolite identification process becomes easier, because everybodys applying a different analytical method, but theyre then trying to identify a subset of all the metabolites in that sample, we only need to identify something once.

Skip to 15 minutes and 35 secondsOnce we know that specific method, then we dont need to identify it again. We might need to confirm it on a regular basis, but we dont need to identify it. Second problem is we dont have all of those authentic chemical standards, so other approaches are required and we have levels of confidence in identifying or annotating metabolites, so ideally we would match it to an authentic chemical standard, we dont at the moment unfortunately for everything because we dont have those authentic chemical standards available. It takes a lot to synthesize them, and a lot of them are not available to buy commercially. So everybodys working on it differently.

Skip to 16 minutes and 9 secondsNew approaches, basically if we knew everything that was present in a blood sample or urine sample or a cell or a tissue sample, before we analyse that data, we would develop an analytical assay, a liquid chromatography mass spectrometry assay for example, which would measure as many of those as we can, and we would know the identity before we collect the data, and thats the ideal goal for metabolomics, we have a list of two thousand, five thousand metabolites that we know are present in serum or in urine, we know the retention time, we know the MS/MS mass spectrum, and therefore we can use that information before we collect the data to develop the most appropriate assay and therefore afterwards quickly we can provide the identification and reach those metabolites as well.

Skip to 16 minutes and 52 secondsSo were not wasting time on the metabolite identification, and we can focus more time on the biological interpretation, which is what were trying to apply in metabolomics. Metabolomics is applied to understand biology within there. So theres a range of other questions, but I think Ive probably spoken long enough, and you might be getting a bit bored of me by now, so again thank you very very much for attending the course, for getting all the way through the course, we know that week four is always a bit more difficult than the first three weeks, its a bit more mathematical and scientific within there, but for everybody to get this far we really thank you for that, and we hope to maybe see you again on some of the other courses within the Birmingham Metabolomics Training Centre.

Skip to 17 minutes and 31 secondsThank you very much.

Course Q & A

Dr Warwick Dunn responds to your comments and question from the course.

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This video is from the free online course:

Metabolomics: Understanding Metabolism in the 21st Century

University of Birmingham