How to measure health improvement? : assessment of subtle shifts in metabolic phenotype
Fazelzadeh, Parastoo - \ 2017
Wageningen University. Promotor(en): A.H. Kersten; J.P.M. van Duynhoven, co-promotor(en): M.V. Boekschoten. - Wageningen : Wageningen University - ISBN 9789463430739 - 187
health promotion - improvement - measurement - metabolic profiling - elderly - obesity - microarrays - rna - peripheral blood mononuclear cells - gezondheidsbevordering - verbetering - meting - metabolische profilering - ouderen - obesitas - microarrays - rna - perifere mononucleaire bloedcellen
Human health is impacted by a complex network of interactions between biological pathways, mechanisms, processes, and organs, which need to be able to adapt to a continuously changing environment to maintain health. This adaptive ability is called ‘phenotypic flexibility’. It is thought that health is compromised and diseases develop when these adaptive processes fail. As the product of interactions between several factors such as genetic makeup, diet, lifestyle, environment and the gut microbiome, the ‘metabolic phenotype’ provides a readout of the metabolic state of an individual. Understanding these relationships will be one of a major challenges in nutrition and health research in the next decades. To address this challenge, the development of high-throughput omics tools combined with the application of elaborate statistical analyses will help characterize the complex relationship of (bio) chemicals in human systems and their interaction with other variables including environment and lifestyle to produce the measured phenotype. An important aim of this thesis was to identify phenotype shifts by looking at effect of prolonged resistance-type exercise training on skeletal muscle tissue in older subjects and the possible shift toward the features of younger subjects as a reference for a healthier phenotype. A second aim was to identify phenotype shifts by looking at the response to a challenge in obese subjects and the possible shift toward lean subjects as a reference for a healthier phenotype.
Chapter 2 and 3 of this thesis show how the significant remaining plasticity of ageing skeletal muscle can adapt to resistance-type exercise training. The data indicate that frail and healthy older subjects have two distinct phenotypes according to the skeletal muscle tissue metabolite profiles and that exercise training shifts aged muscle towards a younger phenotype. We showed that the effect of exercise on amino acid derived acylcarnitines (AAAC’s) in older subjects points towards decreased branched chain amino acid catabolism, likely due to compromised activation of the branched chain α-keto acid hydrogenase (BCKDH) complex. Furthermore, we found that the protocadherin gamma gene cluster might be involved in aged-muscle denervation and re-innervation. Finally, plasma was found to be a poor indicator of muscle metabolism, emphasizing the need for direct assessment of metabolites in muscle tissue.
Chapter 4 of this thesis examines whether a mixed meal challenge response provides a readout for a shift in phenotype upon weight loss in obese male subjects. We concluded that weight loss moderately affects the mixed meal challenge response of both plasma metabolome and transcriptome of peripheral blood mononuclear cells in obese subjects. Measurements at the fasted and postprandial state also provide us with a different type of information.
In Chapter 5 it is demonstrated that the global testing of pathways could provide a concise summary of the multiple univariate testing approach used in Chapter 4. In Chapter 6 it is discussed how the findings of this thesis increase our understanding of how to measure phenotypic flexibility as a proxy of health. In this thesis it is shown that the correlations between tissue and plasma metabolites are rather weak, emphasising the need to perform organ-specific studies. Availability of less invasive/painful sampling techniques and the use of small amounts of tissue would enable larger scale human studies on adipose tissue and skeletal muscle to more accurately define phenotypical shifts due to diet or lifestyle interventions. With respect to the assessment of phenotypical flexibility by omics approaches, significant complications can be expected in trying to relate plasma metabolism to PBMC gene expression. Organ-focussed approaches that integrate multiple omics levels using system biology approaches are considered to be a lot more promising.
Isolation, characterization and engineering of Bacillus smithii : a novel thermophilic platform organism for green chemical production
Bosma, E.F. - \ 2015
Wageningen University. Promotor(en): Willem de Vos; John van der Oost, co-promotor(en): Richard van Kranenburg. - Wageningen : Wageningen University - ISBN 9789462575073 - 220
bacillus smithii - bacillus (bacteria) - biobrandstoffen - chemicaliën uit biologische grondstoffen - thermofielen - metabolische profilering - genoomanalyse - mutaties - isolatie - bioengineering - karakterisering - bacillus smithii - bacillus (bacteria) - biofuels - biobased chemicals - thermophiles - metabolic profiling - genome analysis - mutations - isolation - bioengineering - characterization
Due to the globally increasing demand for chemicals and fuels and the high environmental impact and limited amount of fossil resources, there is a growing interest in green chemicals and fuels derived from renewable resources. As described in Chapter 1, one of the most feasible alternatives on the short term is microbial conversion of the sugars in biomass to fuels and chemicals in a biorefinery. To be economically and ethically feasible, non-food biomass should be used as a resource, which is often difficult with currently used production organisms. Also, to be economically feasible, the costs of green chemicals and fuels need to be further reduced to be below the costs of products based on fossil resources. To do so, other organisms than the currently most-used platform organisms such as Escherichia coli and Saccharomyces cerevisiae should be used. Ideally, this alternative organism is genetically accessible, has high productivity, titre and yield, is flexible in carbon source, robust, moderately thermophilic, acidophilic, facultatively anaerobic and has little nutritional requirements. The organisms that come closest to these criteria are thermophilic bacilli, which form a diverse class of organisms in the family of Bacillaceae. This thesis describes the isolation, characterization and metabolic engineering of Bacillus smithii, a novel potential thermophilic platform organism.
Chapter 2 provides more detail on the use of thermophilic microorganisms as platform organisms for green chemical production in a biorefinery concept. As commercially available enzyme mixtures used in the simultaneous saccharification and fermentation (SSF) of biomass have their optimum temperature around 50-60°C, using a moderately thermophilic organism would reduce the costs of the SSF process compared to when using mesophiles by reducing the amount of required enzyme. Also, thermophilic processes are less prone to contaminations, and substrate and product solubility are increased. Several successful examples of the application of facultatively anaerobic thermophiles for green chemical production from lignocellulose in an SSF setting are for example Bacillus coagulans for lactic acid production and Bacillus licheniformis for 2,3-butanediol production. However, whereas strongly developed genetic toolboxes are available for current mesophilic production organisms, these tools are still in their infancy for thermophilic organisms. Such tools are required to optimize production and to study metabolism. Thermophilic organisms show a wide variety in metabolism and in many cases the metabolism of these organisms is still poorly understood, hampering full optimization. Chapter 2 furthermore provides an overview of transformation, integration and counter-selection methods currently used for thermophiles. Although several deletion mutants have been constructed using these methods, not all of them are entirely markerless and most are not suited as high-throughput engineering tools, stressing the need for further research in this area.
Despite several facultatively anaerobic thermophiles being described as genetically accessible, this feature is still one the major bottlenecks in developing these organisms into platform organisms. Therefore, in Chapter 3, we set out to isolate a facultatively anaerobic, moderately thermophilic bacterium that was genetically accessible and produced high titers of organic acids. A total of 267 strains of different thermophilic bacilli species were isolated from compost and screened for C5 and C6 sugar utilization and acid production. The 44 best strains were screened for genetic accessibility via electroporation. Only 3 strains tested positive for this, namely Geobacillus thermodenitrificans strains ET 144-2 and ET 251 and B. smithii strain ET 138. In subsequent evaluations in lab-scale bioreactors at 55°C and pH 6.5 on glucose, the two G. thermodenitrificans strains performed poorly whereas B. smithii performed well with high titers, yields and productivity of mainly lactate. In similar lab-scale reactors, this strain also performed well on xylose and at pH 5.5 and was still able to perform for 48 at pH 4.5. The electroporation protocol for this strain was optimized, resulting in a maximum efficiency of 5x103 colonies per µg plasmid pNW33n. Two other B. smithii strains, among which the type strain DSM 4216T, were also shown to be transformable with pNW33n. This is the first time that genetic accessibility is described for B. smithii and it is the first step towards developing it into a platform organism, for which it appears to be suitable based on its efficient C5 and C6 sugar utilization and acid production profile.
In order to become a platform organism and to study its atypical metabolism, a genetic toolbox needs to be established for B. smithii. Chapter 5 describes the development of a markerless gene deletion method for B. smithii. For strains ET 138 and DSM 4216T, the ldhL gene was markerlessly removed via double homologous recombination using plasmid pNW33n. Despite the replicative nature of this plasmid at 55°C, mixtures of single and double crossovers were readily obtained. A pure double crossover deletion mutant was obtained after several transfers on a more defined medium containing acetate or lactate and PCR-based screenings. To eliminate the possibility of mixed genotypes, we subsequently developed a lacZ-counter-selection system, which is based on the toxicity of high X-gal concentrations in the presence of the plasmid-encoded lacZ gene. Using this method, the sporulation-specific sigma factor sigF and pyruvate dehydrogenase complex E1-α pdhA were consecutively removed from the B. smithii ET 138 genome in a markerless way. An initial evaluation of the growth and production profiles of the mutant strains in tubes showed that removal of the ldhL gene eliminates l-lactate production and causes a severe decrease in anaerobic growth and production capacities. B. smithii mutants lacking the sigF gene were unable to sporulate and removal of the pdhA gene eliminated acetate production and rendered the strains auxotrophic for acetate.
Systematic metabolite annotation and identification in complex biological extracts : combining robust mass spectrometry fragmentation and nuclear magnetic resonance spectroscopy
Hooft, J.J.J. van der - \ 2012
Wageningen University. Promotor(en): Raoul Bino; Sacco de Vries, co-promotor(en): Jacques Vervoort; Ric de Vos. - S.l. : s.n. - ISBN 9789461732347 - 256
metabolieten - metabolomica - massaspectrometrie - kernmagnetische resonantiespectroscopie - metabolische profilering - metabolische fingerprinting - metabolites - metabolomics - mass spectrometry - nuclear magnetic resonance spectroscopy - metabolic profiling - metabolic fingerprinting
Detailed knowledge of the chemical content of organisms, organs, tissues, and cells is needed to fully characterize complex biological systems. The high chemical variety of compounds present in biological systems is illustrated by the presence of a large variety of compounds, ranging from apolar lipids, semi-polar phenolic conjugates, toward polar sugars. A molecules’ chemical structure forms the basis to understand its biological function. The chemical identification process of small molecules (i.e., metabolites) is still one of the major focus points in metabolomics research. Actually, no single analytical platform exists that can measure and identify all existing metabolites. In this thesis, two analytical techniques that are widely used within metabolite identification studies have been combined, i.e. mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR). MS was used to ionize the metabolites and to record their molecular weight and to provide substructure information based on fragmentation in the mass spectrometer. NMR gave the comprehensive structural information on the chemical environment of protons and their linkage to other protons within the molecule. The additional structural information as compared to MS is at the cost of an increased amount of compound needed for NMR detection and spectra generation. Here we combined both analytical methods into a liquid chromatography (LC)-based platform that concentrated compounds based on their specific mass; thereby providing a direct link between MS and NMR data. Another platform was developed that generated robust multistage MSn data, i.e., the systematic fragmentation of metabolites and subsequent fragmentation of resulting fragments.
This thesis aims to accelerate metabolite identification of low abundant plant and human derived compounds by following a systematic approach. The acquired structural information from MSn and 1D-1H-NMR spectra resulted in the complete elucidation of phenolic metabolites in microgram scale from both plant and human origin.
In the chapter 1, the analytical techniques and terms used throughout the thesis are introduced. The second chapterdescribes how a high mass resolution MSn fragmentation approach was tested in both negative and positive ionization modes for differentiation and identification of metabolites, using a series of 121 polyphenolic molecules. An injection robot was used to infuse the reference compounds one by one into a hybrid mass spectrometer, combining MSn possibilities with accurate mass read-out. This approach resulted in reproducible and robust MSn fragmentation trees up to MS5, which were differential even for closely related compounds. Accurate MSn-based spectral trees were shown to be robust and powerful to distinguish metabolites with similar elemental formula (i.e. isomers), thereby assisting compound identification and annotation in complex biological samples. In the third chapter, we tested the annotation power of this spectral tree approach for annotation of phenolic compounds in crude extracts from Lycopersicum esculentum(tomato) and the model plant Arabipopsis thaliana. Partial MSn spectral trees were generated directly after chromatographic elution (LC-MSn). Detailed MSn spectral trees could be recorded with the use of a collector/injector robot.We were able to discriminate flavonoid glycosides based on their unique MSn fragmentation patterns in either negative or positive ionization mode. Following this approach, we could annotate 127 metabolites in the tomato and Arabidopsis extracts, including 21 novel metabolites. The good quality MSn spectral trees obtained can be used to populate MSn databases and the protocols to generate the spectral trees are a good basis to further expand this database with more diverse compounds.
Chapter 4 then describes how an automated platform, coupling chromatography with MS and NMR (LC-MS-solid phase extraction-NMR), was developed that can trap and transfer metabolites based on their mass values from a complex biological extract in order to obtain NMR spectra of the trapped LC-MS peak, out of minute amounts of sample and analyte. Extracts from tomatoes modified in their flavonoid biosynthesis pathway were used as proof of principle for the metabolite identification process. This approach resulted in the complete structural elucidation of 10 flavonoid glycosides. This study shows that improving the link between the mass signals and NMR peaks derived from the selected LC-MS peaks decreases the time needed for elucidation of the metabolite structures. In addition, automated 1D-1H-NMR spectrum fitting of the experimental data obtained in this study using the PERCH NMR software further speeded up the candidate rejection process.
Chapter 5 illustrates how the two developed analytical platforms could be used for the successful selection, annotation, and identification of 177 phenolic compounds present in different extracts of Camellia sinensis, i.e. green, white, and black tea extracts, including the full identification of microgram amounts of complex acylated conjugates of kaempferol and quercetin. Principal component analysis based on the relative abundance of the annotated phenolic compounds in 17 commercially available black, green and white tea products separated the black teas from the green and white teas, thereby illustrating the differential phenolic metabolite contents of black tea as compared to green and white teas. The change in phenolic profiles reflects the polymerization reactions occurring upon transformation of green tea into black tea. This study shows that the combined use of MSn spectral trees and LC-MS-solid phase extraction-NMR leads to a more comprehensive metabolite description thereby facilitating the comparison of tea and other plant samples.
In chapter 6, we aimed to structurally elucidate and quantify polyphenol-derived conjugates present in the human body by studying the urinary excretion of these conjugates.We applied a combination of a solid phase extraction preparation step and the two HPLC-coupled analytical platforms as described in chapters 2 and 3. This analytical strategy resulted in the annotation of 138 urinary metabolites including 35 completely identified valerolactone conjugates. These valerolactones are microbial break-down products of tea phenols. NMR predictions of glucuronidated and sulphonated core metabolites were performed in order to confirm the NMR peak assignments on the basis of 1D-1H-NMR data only. In addition, 26 hours quantitative excretion profiles for certain valerolactone conjugates were obtained using diagnostic proton signals in the 1D-1H-NMR spectra of urine fractions.
In the seventh chapter, the current state of metabolite identification and expected challenges in the structural elucidation of metabolites at (sub)microgram amounts are discussed. The work in this thesis and of other groups working on the hyphenation of MS and NMR shows that the complete de novo identification of microgram amounts and even lower of compound is feasible by using MS guided solid phase extractiontrapping in combination with 1D-1H-NMR or UPLC-TOF-MS isolation followed by capillary NMR. Semi-automated annotation of compounds based on their MS and NMR features is now feasible for some well studied compound classes and groups.
Altogether, the developed platforms yield new and improved insights in the phenolic profiles of well-studied plants as well as a comprehensive picture of the metabolic fate of green tea polyphenols upon intake in the human body. The followed metabolite identification strategy is useful for other studies that aim to elucidate bioactive compounds, especially when only small sample volumes are available. This thesis also contributes to the acquisition of good quality data for metabolite identification by acquiring robust MSn fragmentation spectra and 1D-1H-NMR spectra of partial purified analytes at microgram scale, which paves the path for further developments in data acquisition and analysis, as well as the unravelling of yet unknown metabolites in a faster, more systematic and automated manner.