Leaf phenolics and seaweed tannins : analysis, enzymatic oxidation and non-covalent protein binding
Vissers, Anne M. - \ 2017
Wageningen University. Promotor(en): H. Gruppen; W.H. Hendriks, co-promotor(en): J.P. Vincken. - Wageningen : Wageningen University - ISBN 9789463432023 - 154
phenols - leaves - seaweeds - tannins - beta vulgaris - laminaria - proteins - catechol oxidase - nuclear magnetic resonance spectroscopy - in vitro - mass spectrometry - browning - fermentation - animal feeding - fenolen - bladeren - zeewieren - tanninen - beta vulgaris - laminaria - eiwitten - catechol oxidase - kernmagnetische resonantiespectroscopie - in vitro - massaspectrometrie - bruinkleuring - fermentatie - diervoedering
Upon extraction of proteins from sugar beet leaves (Beta vulgaris L.) and oarweed (Laminaria digitata) for animal food and feed purposes, endogenous phenolics and proteins can interact with each other, which might affect the protein’s applicability. Sugar beet leaf proteins might become covalently modified by phenolics through polyphenol oxidase (PPO) activity. Oligomeric phenolics from seaweed (so-called phlorotannins (PhT)) might bind non-covalently to protein. The first aim of this thesis was to study factors involved in protein modification by phenolics. The second aim was to investigate the effect of PhT supplementation to feed on in vitro ruminal fermentation.
Besides PPO activity and the amount of low molecular weight phenolic substrates present, brown colour formation in sugar beet leaves was dependent on the amount of phenolics, which do not serve as a substrate of PPO. These non-substrate phenolics can engage in browning reactions by oxidative coupling and subsequent coupled oxidation of the products formed. Similar reactions might also be involved in covalent protein modification by phenolics, and therewith protein properties.
Nanoparticle diffusometry in hydrogels
Kort, D.W. de - \ 2016
Wageningen University. Promotor(en): John van Duynhoven, co-promotor(en): Henk van As. - Wageningen University - ISBN 9789462577459 - 178
nanotechnology - particles - gels - diffusion - nuclear magnetic resonance spectroscopy - rheology - nanotechnologie - deeltjes - gels - diffusie - kernmagnetische resonantiespectroscopie - reologie
In order to understand food product functionality such as elastic and ﬂow behavior and mass transport properties, one ﬁrst has to understand the multi-length-scale structure of the material. The aim of this work is to explore novel methodologies to study and characterize multi-length-scale structures of food hydrogels under static and dynamic conditions. The focus lies on hydrogels comprising polysaccharides, because they show a rich variation in elastic and ﬂow behavior.
The largest part of the thesis focuses on the use of nanoparticles (3–30 nm diameter) that are dissolved into the water phase of hydrogels, and whose mobility is reduced due to the presence of the polymer network. This retardation of nanoparticle self-diﬀusion in hydrogels relative to self-diﬀusion in neat water can be used to infer structural information about the microstructure of the polymer network.
In chapter 2, an in-depth review of existing literature on this method, known as “nanoparticle diﬀusometry”, is provided, with an emphasis on physical models of self-diﬀusion in polymer gels and applications in food gels. In that chapter, we distinguish between (1) nanoparticle diﬀusion in (heterogeneous) polymer gels and (2) nanoparticle diﬀusion in solutions of (semi)ﬂexible polymers. We adhere to this categorization throughout the rest of the thesis.
In chapters 3 and 4 we ﬁrst describe the design and manufacturing of tailor-made nanoparticles that are functionalized with spectroscopic labels, and the implementation of pulsed-ﬁeld gradient (PFG) NMR and optical spectroscopy toolboxes for nanoparticle diﬀusometry. We then use these toolboxes to measure nanoparticle self-diﬀusion in heterogeneous κ-carrageenan (a polysaccharide) gels. These experiments reveal bimodal nanoparticle self-diﬀusion (i.e., there are two nanoparticle fractions with diﬀerent diﬀusion coeﬃcients) as previously observed in these gels by Lorén et al. The results suggest that the sub-micron structure of these gels is heterogeneous with a wide distribution of pore sizes at the sub-micron scale, leading to “sieving” of nanoparticles resulting in the observation of bimodal self-diﬀusion.
This hypothesis is further explored in chapter 5, where besides PFG NMR and optical spectroscopy, Overhauser dynamic nuclear polarization (ODNP)-enhanced NMR spectroscopy is employed. This method can determine the local viscosity of water surrounding the two fractions of particles. It turns out that the particle fraction with the lower apparent diﬀusion coeﬃcient is in fact trapped in small, nanoscopic interstitials within the gel. The ODNP NMR experiments show that the viscosity of water surrounding the trapped particles is signiﬁcantly lower than the viscosity within the larger interstitials.
Chapter 6 describes a study of nanoparticle diﬀusion in solutions of poly(ethylene glycol), a ﬂexible polymer with well deﬁned compositions and chain lengths. We use scaling laws to understand the relation between macroviscosity and “microviscosity” as apparent from the nanoparticle diﬀusivity. We show that the particles probe (near-)macroviscosity only if their size is larger than the size of the PEG polymer coils.
Another topic of this thesis is a study of the behavior of food hydrogels under dynamic conditions. To this end we use rheo-MRI velocimetry, which allows us to study the complex shear ﬂow behavior of hydrogels that (per deﬁnition) have a yield stress. In chapter 7, we ﬁrst employ nanoparticle diﬀusometry to study the sub-micron structure of dispersions of rigid cellulose microﬁbrils in the presence of carboxymethyl cellulose. Carboxymethyl cellulose is a charged cellulose derivative that succeeds to disperse the aggregation-prone cellulose microﬁbrils homogeneously at the sub-micron scale. Rheological characterization shows that the resulting dispersions are thixotropic yield-stress ﬂuids. The ﬂow properties of such ﬂuids are well understood, but rheo-MRI experiments show that shear ﬂow of apparently homogeneous cellulose dispersions does not resemble the ﬂow behavior of typical thixotropic yield-stress ﬂuids. We explain the diﬀerences by using a ﬂuidity model to show that persistent micron-scale heterogeneity still dominates the ﬂow behavior.
Supramolecular nanoparticle interactions and biomolecule detection
Oikonomou, M.E. - \ 2016
Wageningen University. Promotor(en): Aldrik Velders. - Wageningen : Wageningen University - ISBN 9789462576605 - 158
nanotechnology - particles - nuclear magnetic resonance spectroscopy - supramolecular chemistry - lectins - interactions - nanotechnologie - deeltjes - kernmagnetische resonantiespectroscopie - supramoleculaire chemie - lectinen - interacties
Manipulating and understanding matter at the nanoscale describes best the interdisciplinary field of nanotechnology. Nanotechnology is entering, a new era, which is described by Jean-Marie Lehn as the era of “complex matter”. Complex matter is the combination of nanomaterials that together give rise to superstructures, “structures beyond nanostructures”. In this thesis, the motivation was to progressively discover and understand molecular interactions that govern nanoscale natural systems and beyond, and the goal was to acquire the ability to design, direct and control complex matter. In this context, supramolecular ligand interactions on nanoparticle surfaces were designed and implemented with an emphasis on biomolecule sensing. Three main topics were addressed:
1. NMR as a tool in Nanotechnology to study reactions and supramolecular interactions between molecules, nanoparticles and biomacromolecules.
2. Supramolecular Orthogonal Interactions at nanoparticle surfaces.
3. Applications of Supramolecular Orthogonal Interactions for biomolecule recognition and sensing.
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.
Functional magnetic resonance microscopy of long- and short-distance water transport in trees
Homan, N. - \ 2009
Wageningen University. Promotor(en): Herbert van Amerongen, co-promotor(en): Henk van As. - [S.l.] : S.n. - ISBN 9789085855071 - 125
kernmagnetische resonantiespectroscopie - waterstroming - xyleemwaterpotentiaal - bomen - sapstroom - spinthout - nuclear magnetic resonance spectroscopy - water flow - xylem water potential - trees - sap flow - sapwood
Due to their long life span, changing climatic conditions are of particular importance for trees. Climate changes will affect the water balance, which can become an important limiting factor for photosynthesis and growth. Long-distance water transport in trees is directly related to the transpiration stream and very sensitive to changes in the soil-plant-atmosphere water continuum. Therefore the study of long distance transport gives information about tree response to changing climatic conditions. Here the dynamic behaviour of water transport processes in trees has been studied by the MRI method, which is a direct and non-invasive tool.. MRI flow imaging has been applied to diffuse- and ring-porous trees to study drought stress and the occurrence of xylem vessel cavitations.
World-wide unique dedicated MRI hardware is described that allows imaging of sap flow in intact trees with a maximal trunk diameter of 4 cm and height of several meters. This setup is used to investigate xylem and phloem flow in an intact tree quantitatively. Flow is quantified in terms of (averaged) velocity, volume flow (flux) and flow conducting area, either in imaging mode or resolved on the level of annual rings.
Results obtained for the same tree, imaged at two different field strengths (0.7 and 3 T), are compared. An overall shortening of observed T2 values is manifest going from 0.7 to 3 T. Although susceptibility artefacts may be present at 3 T, the results are still reliable and the gain in sensitivity due to the higher magnetic field strength results in shorter measurement time (or a better spatial resolution or a higher signal to noise ratio) with respect to the 0.7 T system. By use of such dedicated hardware xylem and phloem flow, and its mutual interaction, can be studied in intact trees in relation to the water balance and in response to environmental (stress) conditions (Chapter 2).
To further investigate the effect of susceptibility artefacts on MRI flow imaging by PFG-STE MRI on 3 T, water flow was studied in a number of model porous media with or without surface relaxation, internal magnetic field inhomogeneities (susceptibility artefacts) and exchange with stagnant water pools, mimicking the tree situation (Chapter 3). In such situations a clear dependence of the flow characteristics on the observation time is demonstrated. The most reliable results are obtained at relatively short observation times. This limits the observation of low flow velocities and the discrimination between flowing and non-flowing water. It is shown that correlated displacement-T2 measurements are available to improve the discrimination of flowing and non-flowing water and can be of help to decide about the functional activity of xylem conduits (Chapter 4). A method that reveals exchange between the flowing and stagnant fractions in the system is presented. Further it is demonstrated how this exchange can be quantified (Chapter 3).
Xylem flow, flow conducting area and water content in the storage pools of sapwood and cambial zone were investigated simultaneously and non-destructively by MRI in diffuse-porous laurel (Laurus nobilis) and viburnum (Viburnum tinus) trees during a drydown period and recovery after watering (Chapter 4). The development of the drought stress was detected by the decrease in average velocity, volume flow and flow conducting area as observed by MRI flow imaging. A decrease in flow conducting area was observed with a delay of one day in comparison to the observed reduction in average velocity and volume flow. The re-watering of the plants resulted in the fast restore of the flow conducting area to the value observed under well watered conditions, demonstrating that if cavitations had been induced they refilled quite fast. In addition, a significant increase in the average velocity and volume flow was observed, but still lower than the original values. Imaging water content in the cambial zone indicated a gradual decrease of the water content, which speeded up during the drought stress. The rate of decrease was dependent of day/night conditions. Watering resulted in the partial restore of water content in this zone. Water content in sapwood showed a clear diurnal variation. The water storage pool in sapwood depleted quickly upon switching on the light, gradually restoring in the afternoon. Drought stress did not change the character of diurnal variation of water content significantly, but it increased the amplitude of the diurnal variation. Re-watering of the tree resulted in a 10% water loss in sapwood. Thus, for the first time the coupling between water floe in xylem vessels and water content in storage pools was demonstrated. The oldest annual ring was rather inactive in long distance water transport. We found that the transport activity of this ring was not sensitive to any environmental change and that the variation of water content in sapwood was uniform in all annual rings
Non-destructive measurements of cavitation were made with MRI to test whether large earlywood vessels of ring-porous xylem are as vulnerable as some standard methods have suggested (Chapter 5). Potted, 3-4 year old Quercus robur L. trees were droughted to water potentials measured with temperature-corrected stem psychrometers. Imaging of (vessel) water content indicated that earlywood cavitation in trunks was not detectible until water potentials dropped below -3 MPa. Most earlywood vessels were cavitated below -4 MPa. Dye perfusions through excised branch segments gave comparable results. Imaging of flow conducting area (FCA) indicated a gradual decline in trunk water conduction that was not solely associated with cavitation, but probably resulted from stomatal closure and too low velocities to be discriminated from non-flowing water. Dye perfusion and FCA indicated a significant portion of earlywood vessels were non-conducting even at the most favorable water potentials. No refilling of embolized vessels was detected in rewatering experiments. Contradictory to the MRI results, standard centrifuge and air-injection methods on Q. robur stem segments indicated complete cavitation at xylem pressures at or below -1 MPa. An artifact in these destructive methods was revealed by experiments on the related species Q. gambelii Nutt. When earlywood vessels became air-filled during collection prior to being refilled in the lab, they became much more vulnerable to cavitation. Residual bubbles left behind in the refilled vessels may be responsible. These results suggest revised protocols for measuring vulnerability curves by destructive methods.
An about linear correlation between water potential and decrease of water content in cambial zone of oak (Quercus robur L.) was observed (Chapter 5).
Metabolomics technologies applied to the identification of compounds in plants : a liquid chromatography-mass spectrometry - nuclear magnetic resonance perspective over the tomato fruit
Moco, S.I.A. - \ 2007
Wageningen University. Promotor(en): Raoul Bino; Sacco de Vries, co-promotor(en): Jacques Vervoort; Ric de Vos. - [S.l.] : S.n. - ISBN 9789085047421 - 222
solanum lycopersicum - tomaten - metabolieten - fytochemicaliën - kernmagnetische resonantiespectroscopie - metabolomica - lc-ms - solanum lycopersicum - tomatoes - metabolites - phytochemicals - nuclear magnetic resonance spectroscopy - metabolomics - liquid chromatography-mass spectrometry
A new era of plant biochemistry at the systems level is emerging in which the detailed description of biochemical phenomena, at the cellular level, is important for a better understanding of physiological, developmental, and biomolecular processes in plants. This emerging field is oriented towards the characterisation of small molecules (metabolites) that act as substrates, products, ligands or signalling entities in cells. This thesis concerns the development and establishment of such metabolomics strategies for screening and identifying metabolites in biological systems. Most technological strategies were applied to the assignment of metabolites from tomato (Solanum lycopersicum) fruit. Tomato was chosen for being a widely consumed crop with nutritional attributes, representing a model for the Solanaceae family. In order to achieve both high coverage of detected metabolites and valuable information for identification purposes, liquid chromatography coupled to mass spectrometry (LC-MS) and nuclear magnetic resonances (NMR) technologies were used. In addition, metabolite databases, based on experimental data (mass-based, in the case of LC-MS and chemical shift-based, in the case of NMR) were initiated, in order to systemize the extensive metabolite information. The chapters in this thesis describe method developments and their applications in plant metabolomics that are also feasible to be implemented on other biological systems. A review on the technologies used for metabolomics with a perspective on compound identification is presented in Chapter 1. In Chapter 2, a robust large scale LC-MS method for the analysis of metabolites in plants is described in detail. It presents a step-by-step protocol with thorough information about the reagents used, sample preparation, instrument setup, methods of analysis and data processing strategies. The described analytical method combines LC with photo diode array (PDA) and MS detection, and allows the analysis of mostly semi-polar secondary metabolites present in plants, such as phenolic acids, flavonoids, glucosinolates, saponins, alkaloids and derivatives thereof. Chapter 3 presents an application of the LC-PDA-MS method for the profiling of metabolites present in tomato fruit. The metabolites putatively identified in this fruit were included in a tomato dedicated-database (the MoTo DB) that is available for public search on the web (see: http://appliedbioinformatics.wur.nl). A comparison between two tomato fruit tissues, peel and flesh, for their metabolite content was made using this MoTo DB. Using the same LC-PDA-MS setup, several different tomato fruit tissues were compared in more detail, along the fruit ripening timeline, in Chapter 4. The presence of tissue-specific metabolites, at determined ripening stages, suggests developmental control of metabolite biosynthesis. Such tissue-specific metabolomics approach may give rise to a biological view over metabolite compartmentalisation. Chapters 5 and 6 describe the implementation of a NMR database for secondary metabolites, mostly including flavonoids, the Flavonoid Database (see: Flavonoid Database under http://www.wnmrc.nl). The acquisition of a large data set of related standard compounds allowed the analysis of shifts in NMR characteristics by the presence of certain functional groups or substituents in the flavonoid backbone. In addition, a 1H NMR-based prediction model was iteratively trained from the acquired experimental data and can be used for the prediction of unknown related molecules. This approach greatly increases the efficiency in the identification of (flavonoid) metabolites. Chapter 7 describes correlations of metabolomics data derived from LC-MS and NMR analyses of a large number of different tomato cultivars. The identification of metabolites is obtained among other available sources, the MoTo DB and the Flavonoid Database. This approach illustrates the complementariness and coincidence of NMR and MS as analytical techniques, applied to the detection of metabolites in tomato fruit. The summarizing discussion and conclusions, sets the work presented in this thesis into a biochemical perspective, and prospects suggestions for the future.
Isolation and Identification of Kairomone(s) in the Daphnia-Scenedesmus System
Holthoon, F.L. van - \ 2004
Wageningen University. Promotor(en): Aede de Groot, co-promotor(en): Teris van Beek; E. van Donk. - Wageningen : S.n. - ISBN 9789085040668 - 154
daphnia - predator prooi verhoudingen - waarschuwingsferomonen - isolatietechnieken - chemische structuur - fractionering - hplc - kernmagnetische resonantiespectroscopie - daphnia - predator prey relationships - alarm pheromones - isolation techniques - chemical structure - fractionation - hplc - nuclear magnetic resonance spectroscopy
Infochemicals play an important role in interactions between living organisms in aquatic environments. Although the presence of these chemical cues is confirmed in more and more systems, the chemical structures of the compounds involved remain predominantly elusive and the identification of these compounds is essential to advance the research on chemical communication. An overview of chemical cues involving Daphnia (either as producer or receiver) is given and the progress towards their isolation and structure elucidation is described (Chapter 1). Most of the research so far has concentrated on the elucidation of kairomones produced by predators of Daphnia (especially Chaoborus and several species of fish). Less study has been devoted to the isolation of the infochemical exuded by Daphnia that causes colony formation in its prey Scenedesmus. One of the main aims of this study was the isolation and identification of this chemical cue. Colony formation in Scenedesmus only occurs when unicellular populations are exposed to either Daphnia or water that had contained Daphnia. It was concluded that the responsible cue had a chemical rather than a mechanical nature, since filtered Daphnia water also showed the colony formation activity. This colony formation was the basis for the development of a bioassay (Chapter 2). A bioassay is a test that is used to measure biological activity (in this case colony formation) of chemical mixtures or biological parameters. Colonies are indicated by high values and single cells are indicated by low values. Unfortunately over time a gradual decline of the difference between negative and positive controls was observed and efforts were undertaken to determine the cause for this decline. Several conditions were investigated (such as time, temperature, algae strain, culture medium, location, incubator, Erlenmeyer size, bacterial growth and microevolution). Additionally some general properties of the kairomone (such as thermal decomposition, biodegradation and concentration) were tested. A correlation between any of the above mentioned factors and the gradual decline of the difference between negative and positive controls was not found. Given that the bioassay was performed under such highly variable and not strictly controlled circumstances, this particular bioassay seems to be rather robust. However this does not defer from the fact that the quality of the bioassay did decline over time. Until the variable is identified that is responsible for the observed decline in difference between positive and negative controls, more care should be taken to standardise as many variables as possible. Despite its drawbacks, a bioassay still remains the best option to guide isolations of bioactive compounds through controlled experiments as long as observed differences are statistically significant. To find the most suitable and practical method for the analysis of Daphnia test water several sample pre-treatment methods were compared (Chapter 3), such as liquid-liquid extraction, solid-phase extraction, stir-bar sorptive extraction, solid-phase disk extraction. A test mixture with ten known natural compounds differing in polarity (log K o/w between -4.34 and 3.70) was used. The best method for small amounts of sample was either 'stirred' SPE or 'cartridge' SPE, but for large amounts of sample (sometimes up to 20 L) 'syringe' SPE was more suited. Consequently an SPE analysis protocol was developed that could elute the active compound in one fraction (Chapter 4). The experiments were performed with different concentrations of organic solvents and different sorbents (endcapped C 18 , MF C 18 , non-endcapped C 18 , C 8 , C 2, CN, ENV + and Oasis ® HLB). Endcapped C 18 was eventually chosen for further experiments (other sorbents did not perform better) and extracted with differing concentrations of methanol in water (50%, 85%) and pure methanol (100%). The chemical cue was most often recovered from the 85% aqueous methanol fraction, which indicates the cue is moderately non-polar.Biological activity was lost when active Daphnia water was partitioned at pH 12.0 against ethyl acetate. The aqueous and organic layer were both inactive, either by inactivation of the kairomone by the basic conditions in the aqueous layer or possibly more than one compound is present with synergistic effects. At lower pH (2.0 and 7.0) biological activity was recovered from the organic layer. This could be an indication that the active compound contains an anionic group. Experiments performed with ion exchange materials (SAX, SCX and Amberlite IRA-400) focused initially on the anion exchanger (SAX). However colony formation activity was recovered from the unretained fraction in contrast to what had been reported previously. This unexpected result prompted extraction with a cation exchanger (SCX). To exclude problems related to pH sensitive silica based sorbents, experiments were repeated on a resin based sorbent (Amberlite IRA-400),however a similar result was obtained as with the SAX sorbent. No satisfying explanation was found for the presence of biological activity in the unretained fractions and absence from the retained fractions, but different counterions on the ion exchangers could play a role. The enriched extracts obtained by SPE (C 18 ) were fractionated by high performance liquid chromatography. One fraction showed a significantly higher biological activity relative to the control ('Fraction C'). Further fractionation yielded three active fractions (C2, C3,C6). This could be an indication that more than one compound is responsible for the activity. Several natural products were biologically inactive when screened in the bioassay. They were, ecdysterone and juvenile hormone III (important hormones in other Crustaceae ), urea (proposed as kairomone in Daphnia - Scenedesmus system ), and geranic acid (reference compound). Although an assumption was made to ignore possible synergistic or additive effects in these experiments, the possibility of synergism or additivity should not be ignored, given that most likely more than one active fraction is present. At this point, due to the lack of reproducible and significant results from bioassay-guided separations, another way to identify possible candidates for the role of kairomone had to be used. Daphnia and control water were first extracted using SPE and then analysed with chromatographic techniques. Chromatograms of biologically active extracts were then compared with chromatograms of non-active control extracts to determine and recognise unique peaks (i.e. peaks only present in active Daphnia test water extracts). In an attempt to maximise the available data on the unknown colony inducing compound(s) several techniques were applied simultaneously, such as gas and liquid chromatography (Chapter 5). Several small unique peaks were recognised in the silylated extracts of Daphnia test water with GC-MS analysis. Some of these were tentatively identified as dodecanol, azelaic acid, sebacic acid and veratroylformic acid, but they did not induce colonisation. HPLC detection was performed not only with ultraviolet spectroscopy but also with evaporative light scattering and by electrospray ionisation-mass spectrometry to avoid overlooking compounds without a UV chromophore. LC analysis on four columns with different packings ensured that peaks were well separated on at least one column. Chromatograms with the best resolution were obtained on a C 18 column with an ACN-H 2 Ogradient and UV detection. High noise levels reduced the usefulness of ELS detection. Several peaks unique to 90% aqueous MeOH extracts of Daphnia test water were detected, but unfortunately not identified. Some of the recognised unique peaks ( B, G,K ) eluted in previously identified active regions ('Fraction C'). Especially peak B ([M-H] ¯ = 752.8 ?,lmax227 nm) was present in high amounts and well separated from neighbouring peaks. Therefore this peak was further analysed by liquid chromatography-nuclear magnetic resonance. Peaks from several extracts were trapped onto one SPE cartridge. This way a sufficient amount of analyte could be transferred into the NMR probe to allow recording of a 1-dimensional 1 H-spectrum. Unfortunately the spectrum did not lead to elucidation of the structure of peak B . One aliquot of this collected fraction was therefore analysed by high-resolution mass spectrometry and liquid-chromatography-quadrupole time-of-flight mass-spectrometry to obtain an accurate mass, while another aliquot was checked for biological activity in a bioassay. Unfortunately analysis with HRMS was unsuccessful and analysis with LC-QTOF has not yet yielded results. The peak with a possible pseudo molecular mass of 752.8 ([M-H] ¯) could not be detected. The other aliquot that was tested for biological activity in the bioassay showed significant differences between the negative control, positive control and peak B . This peak could therefore play a role in the induction of colonies in Scenedesmus , although it is still unclear whether it acts alone. Should peak B prove to be (partly) responsible for colony formation in Scenedesmus then the most important objective of this study has been partly reached, namely the isolation of kairomone(s) in the Daphnia - Scenedesmus system. This information will enable and facilitate research into the other objectives.
Wet chemical and phosphorus-31 nuclear magnetic resonance analysis of phosphorus speciation in a sandy soil receiving long-term fertilizer or animal manure applications
Koopmans, G.F. ; Chardon, W.J. ; Dolfing, J. ; Oenema, O. ; Meer, P. van der; Riemsdijk, W.H. van - \ 2003
Journal of Environmental Quality 32 (2003). - ISSN 0047-2425 - p. 287 - 295.
bodemchemie - fosfor - grondanalyse - zandgronden - uitspoelen - chemische speciatie - kernmagnetische resonantiespectroscopie - sandy soils - soil chemistry - leaching - phosphorus - soil analysis - chemical speciation - nuclear magnetic resonance spectroscopy - northern vancouver-island - organic phosphorus - p-31 nmr - podzolic soils - forms - spectroscopy - extraction - netherlands - chemistry - fractions
In areas under intensive livestock farming and with high application rates of animal manure, inorganic and organic phosphorus (P) may be leached from soils. Since the contribution of these P compounds to P leaching may differ, it is important to determine the speciation of P in these soils. We determined the effect of various fertilization regimes on the P speciation in NaOH–Na2EDTA (ethylenediaminetetraacetic acid) and water extracts of acidic sandy soil samples from the top 5 cm of grassland with wet chemical analysis and 31P nuclear magnetic resonance (NMR) spectroscopy. These soils had been treated for a period of 11 years with no fertilizer (control), N (no P application), N–P–K, or different animal manures. Inorganic P was highly elevated in the NaOH–Na2EDTA extracts of the soils amended with N–P–K or animal manures, while organic P increased only in the soil treated with pig slurry. Water-extractable P showed a similar trend. As indicated by 31P NMR, orthophosphate monoesters were the main organic P compounds in all soils. Our results suggest that long-term applications of large amounts of P fertilizer and animal manures caused an accumulation of inorganic P, resulting in an increase of the potential risk related to mobilization of inorganic P in the top 5 cm of these soils.
Characterisation of complex xylo-oligosaccharides from xylan rich by-products
Kabel, M.A. - \ 2002
Wageningen University. Promotor(en): A.G.J. Voragen; H.A. Schols. - S.l. : S.n. - ISBN 9789058086969 - 128
xylaan - oligosacchariden - hydrolysaten - karakterisering - bijproducten - hout - eucalyptus - tarwezemelen - maïsspillen - brouwgranen - gerst - verteerbaarheid - chromatografie - massaspectrometrie - kernmagnetische resonantiespectroscopie - xylan - oligosaccharides - hydrolysates - characterization - byproducts - wood - eucalyptus - wheat bran - maize cobs - brewers' grains - barley - digestibility - chromatography - mass spectrometry - nuclear magnetic resonance spectroscopy
Hydrolysates obtained by hydrothermal treatment of four xylan rich by-products (wheat bran, brewery's spent grain, corn cobs and Eucalyptus wood) were characterised. Depending on the feedstock material studied, the xylan originally present differed in substitution with arabinose, 4- O -methylglucuronic acid and O -acetyl substituents. Due to a partial release of the various substituents and depolymerisation of the xylan by the hydrothermal treatments performed, a wide variety of differently substituted XOS and xylan-fragments were obtained.
High performance anion-exchange chromatography (HPAEC), reversed phase (RP)-high performance liquid chromatography (HPLC), mass spectrometry (MS), NMR spectroscopy, RP-HPLC-MS and RP-HPLC-NMR showed to be very useful for the separation and characterisation of the detailed structures of the substituted XOS.
The differently substituted XOS in the hydrolysates of brewery's spent grain and Eucalyptus wood were separated by anion-exchange and size-exclusion chromatography and characterised in more detail. The XOS in the brewery's spent grain hydrolysate included mainly xylan-fragments substituted with arabinoses and XOS containing only few substituents. Furthermore, arabinoxylan-fragments having O -acetyl substitution were present, suggesting the presence of O -acetyl in cereal arabinoxylans. The Eucalyptus wood hydrolysate contained mainly linear XOS, O -acetylated XOS and ( O -acetylated) XOS substituted with one or two 4- O -methylglucuronic acid(s). Additionally, a series of XOS containing both 4- O -methyl-glucuronic acid and a hexose, most likely galactose, was identified.
Studies on the pro-oxidant chemistry of flavonoids
Awad, H.M. - \ 2002
Wageningen University. Promotor(en): I.M.C.M. Rietjens; P.J. van Bladeren; J. Vervoort. - S.l. : S.n. - ISBN 9789058085887 - 133
flavonoïden - oxidatiemiddelen - antioxidanten - quercetine - structuuractiviteitsrelaties - hplc - kernmagnetische resonantiespectroscopie - massaspectrometrie - flavonoids - oxidants - antioxidants - quercetin - structure activity relationships - hplc - nuclear magnetic resonance spectroscopy - mass spectrometry
There is currently much interest in the development of functional foods aiming at the prevention of the development of some diseases, for example cancer, by the introduction of selected natural substances at elevated levels into the diet. The rationale for this approach is based especially on epidemiological data that indicate that food items containing such chemicals may reduce the risk of these diseases in humans. Epidemiological studies indicate, for example, that diets rich in fruit and vegetables protect against a variety of diseases, including heart diseases and certain forms of cancer. However, identification of the actual ingredient in a specific diet responsible for the beneficial health effects remains an important bottleneck for translating observational epidemiology to development of a functional food ingredient. The protection against cancer afforded by fruit and vegetables has been attributed to antioxidant micronutrients such as vitamin C, beta-carotene and vitamin E, which may act at many sites, including the stomach, intestine, lung and bladder. However, present scientific attention is focusing as well on the significance of other minor dietary components, notably the flavonoids as protectants against disease. Flavonoids are widespread in nature and are found in considerable quantities in fruits, vegetables, seeds, peel and tubers. The average Western diet may provide up to 1 g of flavonoids per day. Numerous in vitro studies show that flavonoids are potent antioxidants and metal chelators. Their potential as anti-inflammatory, antiallergic and antiviral compounds has also attracted attention. These studies provide the basis for the present rapidly increasing interest for the use of flavonoids as functional food ingredients. As a result increased human exposure to flavonoids can be expected in the near future. In shops and at the internet, food and food supplements based on (iso)flavonoids as functional ingredients are marketed. This, although hard scientific data supporting the health claims as well as data allowing a balanced risk-benefit evaluation are lacking. For flavonoids increased future human exposure regimens induce the question on their pro-oxidant chemistry. There is considerable evidence that some flavonoids are mutagenic in both bacterial and mammalian experimental systems. A high incidence of gastric cancer in some human populations has been linked to consumption of wine containing potentially mutagenic flavonoids (Tamura et al. , Proc. Natl. Acad. Sci. USA. 77, 4961-4965, 1980, Hoey et al. , Am. J. Epidemiol., 113, 669-974, 1981). Relatively little is understood about either the toxicity or protection afforded by flavonoids in humans.
Since flavonoid quinone/quinone methides have been suggested as the major metabolites responsible for the possible pro-oxidant toxicity and mutagenicity of flavonoids, characterisation of flavonoid quinone chemistry is of importance. However, little information is available on the structure and reactivity of these flavonoid oxidation products. Therefore, the objective of this thesis was to investigate the pro-oxidant chemistry of flavonoids and to perform structure activity studies on the chemical behaviour of 3',4'-dihydroxyflavonoids with special emphasis on the nature and reactivity of the quinone/quinone methide type metabolites formed. Using the GSH trapping method, HPLC, LC/MS, MALDI-TOF, 1H NMR, 13C NMR and quantum mechanical computer calculations the quinone/quinone methide chemistry of a series of 3',4'-dihydroxyflavonoids could be characterised.
The results provide insight in structure-activity-relationships for the pro-oxidant chemistry of these electrophilic quinone/quinone methide flavonoid metabolites. The results obtained also reveal an unexpected pH-dependent electrophilic behaviour of B ring catechol flavonoids. Furthermore the results of this thesis also reveal, for the first time, evidence for the pro-oxidative chemistry of quercetin in a cellular in vitro model. The formation of these glutathionyl-flavonoid adducts provides evidence for the actual pro-oxidative formation of reactive quinone type metabolites from B ring catechol flavonoids in the selected cellular in vitro model using melanoma cells. Oxidation of the catechols to quinones and their isomeric quinone methides generates potent electrophiles that could alkylate DNA. Interestingly, the structural requirements essential for good antioxidant activity match the requirements essential for pro-oxidant action and quinone methide formation. Altogether, the pro-oxidant behaviour of flavonoids and their quinone/quinone methides are far from straight forward and need to be re-evaluated especially in the framework of the risk-benefit evaluation of the use of these flavonoids as functional food ingredients and/or food supplements.
Er is momenteel veel interesse voor de ontwikkeling van functionele voedingsmiddelen (functional foods), met als doel het voorkomen van het ontstaan van ziekten zoals bijvoorbeeld kanker, via het in verhoogde mate introduceren van geselecteerde natuurlijke bestanddelen in het dieet. De basis voor deze aanpak wordt momenteel met name gevonden in epidemiologische studies die laten zien dat diëten rijk aan specifieke voedselcomponenten of ingrediënten de kans op bepaalde ziekten bij de mens verlagen. Zo geven epidemiologische studies bijvoorbeeld aan dat diëten die rijk zijn aan fruit en groenten beschermen tegen een aantal ziekten zoals hartziekten en bepaalde vormen van kanker. Echter, het identificeren van de belangrijke ingrediënten in het betreffende dieet die het gezondheidsbevorderende effect tot stand brengen is een knelpunt voor het vertalen van de resultaten uit de epidemiologie naar de ontwikkeling van een functioneel voedingsingrediënt.
De bescherming tegen kanker door groenten en fruit is toegeschreven aan antioxidanten zoals vitamine C, beta-caroteen en vitamine E, die op vele plaatsen in het lichaam, zoals de maag, darmen, long en de blaas actief zijn. Wetenschappelijk wordt momenteel veel aandacht besteed aan het mogelijke belang van andere belangrijke dieet componenten, zoals flavonoïden, als beschermende ingrediënten tegen ziekte. Flavonoïden komen in de natuur veel voor, en worden met name in hoge concentraties gevonden in fruit, groenten, knollen en zaden. Het gemiddelde Westerse dieet bevat ongeveer 1 gram aan flavonoïden per dag.
Vele in vitro studies tonen aan dat flavonoïden goede antioxidanten en metaal chelatoren zijn. Daarnaast hebben ze anti-inflammatoire, anti-allergische en anti-virale eigenschappen die van belang worden geacht. Deze bevindingen verschaffen de basis voor de momenteel snel groeiende interesse om flavonoïden te gebruiken als functionele voedingsingrediënten. Als gevolg hiervan zou er in de nabije toekomst een toename in de opname van flavonoïden via het dieet verwacht kunnen worden. In winkels en via het internet worden voedingsmiddelen en voedingssupplementen gebaseerd op (iso)flavonoïden als functionele voedingsingrediënten verkocht. Dit, terwijl zowel de wetenschappelijke onderbouwing voor de gezondheidsclaims als gegevens die een gebalanceerde "risk-benefit" analyse mogelijk maken, nog ontbreken. In het geval van verhoogde toekomstige blootstelling van mensen aan flavonoïden worden voor de risk-benefit evaluatie vragen van belang rond hun mogelijk pro-oxidatieve chemisch gedrag. Er zijn aanwijzingen dat sommige flavonoïden mutageen zijn in zowel bacteriële als zoogdier in vitro test systemen. Een verhoogde mate aan maagkanker in bepaalde humane populaties is in verband gebracht met de consumptie van wijn met daarin mogelijk mutagene flavonoïden (Tamura et al. , Proc. Natl. Acad. Sci. USA. 77, 4961-4965, 1980, Hoey et al. , Am. J. Epidem., 113, 669-974, 1981). Alles samenvattend is er eigenlijk weinig bekend van de schadelijke maar ook van de gezondheidsbevorderende effecten van flavonoïden.
Omdat flavonoid chinon/chinon methides genoemd zijn als de belangrijkste metabolieten die verantwoordelijk zouden zijn voor de mogelijke pro-oxidatieve toxiciteit en mutageniteit van flavonoïden, is karakterisering van deze pro-oxidant chemie van flavonoïden van belang. Echter er is weinig bekend over de structuur en de reactiviteit van deze flavonoid oxidatie producten. Daarom was het doel van deze studie de pro-oxidant chemie van flavonoïden te onderzoeken en een structuur-activiteits studie uit te voeren naar het chemische gedrag van 3',4'-dihydroxyflavonoïden. Daarbij werd speciale aandacht besteed aan de aard en reactiviteit van de gevormde chinon/chinon methide metabolieten. Met behulp van de GSH-trapping methode, HPLC, LC/MS, MALDI-TOF, 1H-NMR, 13C-NMR en kwantum-chemische computerberekeningen kon de chinon/chinon methide chemie van een serie 3',4'-dihydroxyflavonoiden gekarakteriseerd worden.
De verkregen resultaten geven inzicht in de structuur-activteits relaties voor de pro-oxidatieve chemie van de electrofiele chinon /chinon methides metabolieten van de flavonoïden. De resultaten laten ook een onverwacht effect zien van de pH op het electrofiele gedrag van de B-ring catechol flavonoïden. Bovendien laten de resultaten van het proefschrift zien dat zelfs onder reducerende omstandigheden in een cellulair in vitro model (melanoma cellen) de pro-oxidatieve chemie van quercetine van belang kan zijn. Met name de vorming van glutathion-flavonoid conjugaten is een bewijs dat in het gekozen cellulaire model de pro-oxidatieve vorming van reactieve flavonoid chinon/ chinon methide metabolieten is opgetreden. Oxidatie van de catecholen naar chinonen en hun isomere chinon methides genereert electrofielen die DNA kunnen alkyleren. Van belang is dat de structurele randvoorwaarden die een flavonoid een goede antioxidant maken gelijk blijken te zijn aan de structurele kenmerken die essentieel zijn voor pro-oxidant gedrag en chinon methide vorming.
Al met al is de pro-oxidant chemie van flavonoïden en van hun chinon /chinon methides verre van recht toe recht aan gebleken en zou de pro-oxidatieve chemie en de toxiciteit van de flavonoïden in het kader van hun gebruik als functional food ingredienten beter onderzocht en afgewogen moeten worden, rekening houdend met hun mogelijk gezondheidsbevorderende effecten.
Elemental quantitation of natural organic matter by CPMAS C-13 NMR spectroscopy
Conte, P. ; Piccolo, A. ; Lagen, B. van; Buurman, P. ; Hemminga, M.A. - \ 2002
Solid state nuclear magnetic resonance 21 (2002). - ISSN 0926-2040 - p. 158 - 170.
bodemchemie - organische stof - chemische analyse - kernmagnetische resonantiespectroscopie - soil chemistry - organic matter - chemical analysis - nuclear magnetic resonance spectroscopy
NMR of porous bio-systems
Snaar, J.E.M. - \ 2002
Wageningen University. Promotor(en): T.J. Schaafsma; H. van As. - S.l. : S.n. - ISBN 9789058087188 - 170
kernmagnetische resonantiespectroscopie - poreus medium - celmembranen - membraanpermeabiliteit - nuclear magnetic resonance spectroscopy - porous media - cell membranes - membrane permeability
The structure and dynamics of water diffusion and -transport at a microscale in heterogeneous porous media have been investigated using various 1H NMR techniques. In particular in biological porous media the dynamics are usually very complex since it is intimately related to the microstructure (close environment). Fortunately, there is an almost endless variety of NMR pulse sequences available for measuring spin density, relaxation, and diffusion, although finding the most suitable sequence for a particular case is not always straightforward.
The main objectives of the research described in this Thesis were:
(i). increasing our understanding of the microdynamics and microstructure of porous media by collecting a maximum of information using NMR relaxation and/or diffusion measurements, in combination with
(ii). designing and testing robust experimental NMR protocols for the simultaneous measurements of diffusion and relaxation, and
(iii). formulating mathematical models, either numerical or analytical, of the dynamic processes being measured.
For a better understanding of NMR principles relevant to the research described in this Thesis and to provide an easy and straightforward access to the NMR-related properties of the systems under investigation, a general theoretical Chapter has been added. This Chapter treats the underlying principles behind various NMR methods to measure molecular motion.
Both plant tissue and model systems, consisting of beds of glass microspheres, have been investigated. Apple parenchyma tissue has been used throughout, and serves as an example of cellular tissue with relatively good cell uniformity. Cellular tissue has water transport through internal structures and semipermeable membranes. In addition, cell walls and internal organelles represent obstructions to water diffusion.
First of all a new method for the simultaneous measurement of T 1 and T 2 relaxation times is introduced. This results in improved accuracy of T 1 measurement, and a demonstration of the coupling of T 1 and T 2 .
The next chapter describes how paramagnetic ions can be used to identify three populations of water in parenchyma apple tissue with different relaxation characteristics, and how the tonoplast permeability can be calculated from the experimental results. The observed relaxation times originate from particular water compartments: the vacuole, the cytoplasm and the cell wall / extracellular space.
Generally, porous biomedia is spatially heterogeneous. As a result the contributions of relaxation and diffusion to the signal amplitude becomes intimately coupled together. This coupling is demonstrated for a system with a simple geometry, where the walls act as relaxation sinks. Analytical expressions have been obtained, and the effect of loss of magnetization at the pore walls on diffusion measurements and q-space microscopy is demonstrated. Methods to obtain correct values for system parameters such as the pore size, the self-diffusion coefficient, the wall relaxation strength, from experimental results are presented.
In order to combine the relaxation time and diffusion measurements various pulse sequences can be applied. Which one to chose depends both on the type of the investigated system and the static magnetic field strength. A thorough analysis of coupled diffusion and relaxation in multi-compartment systems such as cellular tissue and porous structures requires numerical solution of the coupled Bloch-Torrey equations for each compartment. The next chapter introduces a numerical cell model whereby each cell is modeled as a set of compartments, separated by membrane barriers of variable permeability and surface relaxation strength. Both the static structure factor of cellular tissue measured with q-space microscopy, and dynamic information is obtained. The coupling of diffusion and spin relaxation is demonstrated using the numerical multi-compartment cell model. Spatially inhomogeneous relaxation is shown to distort the apparent static and dynamic structure factors. Changes in compartment morphology alter the coupling of relaxation and diffusion.
Following this, results of preliminary experiments on heterogeneous and metabolically active plant tissue are discussed. Slow physiological changes in the tissue is followed using relaxation time measurements. The response to both growth and fungal infection are compared with predictions using numerical cell models.
As an example of non-biological porous media beds of glass microspheres have been studied. First of all the effects of changes in air-water distributions with lowering water content in various monodisperse glass bead beds on relaxation time distributions are discussed. Pores in a randomly packed bed of spheres are not spherical, however, but consists of distorted octahedral and tetrahedral pores, and triangular shaped "throats" having six, four, and three "corners," respectively (points of contact between glass spheres). Relaxation measurements yield both the mean pore size as well as the detailed pore geometry. Changes in relaxation time distributions are compared with various models of pore emptying. This is followed by a description of combined diffusion and relaxation measurements using a single, robust NMR protocol for microstructural determination of monodisperse glass bead beds. A decrease in water content changes the magnitude of local susceptibility induced field gradients in the pores. These gradients make the differentiation of pore size and geometry possible and increase the dynamic information obtainable. Also, the remarkable susceptibility effects in the NMR analysis of pore distribution are discussed.
Nuclear magnetic resonance imaging of water motion in plants
Scheenen, T.W.J. - \ 2001
Wageningen University. Promotor(en): T.J. Schaafsma; H. van As. - S.l. : S.n. - ISBN 9789058084750 - 133
plantenfysiologie - plant-water relaties - waterstroming - stamafstroming - cavitatie - embolie - diffusie - kernmagnetische resonantiespectroscopie - kernmagnetische resonantie - vaten (plantenweefsel) - xyleem - plant physiology - plant water relations - water flow - stemflow - cavitation - embolism - diffusion - nuclear magnetic resonance spectroscopy - nuclear magnetic resonance - vessels - xylem
This Thesis treats one of the new techniques in plant science i.e. nuclear magnetic resonance imaging (NMRi) applied to water motion in plants. It is a challenge, however, to measure this motion in intact plants quantitatively, because plants impose specific problems when studied using NMRi. At high magnetic field strength air-filled intercellular spaces in the plant tissue cause susceptibility-related local magnetic field inhomogeneities, which are much smaller at low magnetic field strength. The inherently low signal-to-noise ratio at low magnetic fields is compensated by the possibility to record a long train of spin-echoes, since generally the spin-spin relaxation time T 2 at low magnetic field is longer than at high magnetic field.
In this Thesis the spin echo train is used to shorten the time to produce an NMR image. As a result, time-dependent flow phenomena can be followed at a physiologically relevant time scale using dynamic NMRi employing either a pulsed field gradient (PFG) spin echo sequence (for fast flow, Chapter 2) or a PFG stimulated echo motion-encoding sequence (for slow flow, Chapter 3). Using the quantification method presented in this Thesis (Chapter 4) a number of flow characteristics can be determined for every pixel in an image of a plant stem:
These flow characteristics, together with the water density (or total amount of water) and the T 2 value per pixel (measured with quantitative T 2 imaging), were studied in the stem of a cucumber plant as a function of the day-night cycle and cooling of the root system. Root cooling results in inhibition of the water uptake and xylem- and phloem transport, and causes severe wilting of the plant leaves. Following root cooling, during recovery of the plant from its wilted state, the T 2 -values of tissue around the vascular bundles strongly decrease, which may indicate an increased membrane permeability for water of the tissue cells in this period (Chapter 5).
During root cooling, large negative pressures in the plant xylem cause cavitations in the vessels, blocking further water transport. In this Thesis the first direct in vivo observations of refilling of cavitated xylem vessels are presented (Chapter 6). This refilling takes many hours and occurs while nearby vessels are under tension and are transporting water. This finding has important implications for the mechanism underlying the refilling process: water entering the refilling vessel must be hydraulically isolated from flowing water in nearby vessels.
The strategy (Chapter 7) and methodology of quantitative flow and T 2 NMR imaging, discussed in this Thesis has opened new ways to find answers to longstanding questions in plant science.
Composition of plant tissues and soil organic matter in the first stages of a vegetation succession
Nierop, K.G.J. ; Lagen, B. van; Buurman, P. - \ 2001
Geoderma 100 (2001). - ISSN 0016-7061 - p. 1 - 24.
organisch bodemmateriaal - plantensuccessie - humus - kernmagnetische resonantiespectroscopie - soil organic matter - plant succession - humus - nuclear magnetic resonance spectroscopy
Quantitative aspects of solid-state 13C-NMR spectra of humic substances from soils of volcanic systems.
Conte, P. ; Piccolo, A. ; Lagen, B. van; Buurman, P. ; Jager, P.A. de - \ 1997
Geoderma 80 (1997). - ISSN 0016-7061 - p. 327 - 338.
vulkanische gronden - andepts - andosols - organische verbindingen - bodem - bodemchemie - kernmagnetische resonantie - kernmagnetische resonantiespectroscopie - volcanic soils - andepts - andosols - organic compounds - soil - soil chemistry - nuclear magnetic resonance - nuclear magnetic resonance spectroscopy
Quantitative differences in evaluating soil humic substances by liquid- and solid-state 13°C-NMR spectroscopy.
Conte, P. ; Piccolo, A. ; Lagen, B. van; Buurman, P. ; Jager, P.A. de - \ 1997
Geoderma 80 (1997). - ISSN 0016-7061 - p. 339 - 352.
organische verbindingen - bodem - bodemchemie - kernmagnetische resonantie - kernmagnetische resonantiespectroscopie - organic compounds - soil - soil chemistry - nuclear magnetic resonance - nuclear magnetic resonance spectroscopy
Displacement imaging in porous media using the line scan NMR technique
Dusschoten, D. van; Noort, J. van; As, H. van - \ 1997
Geoderma 80 (1997). - ISSN 0016-7061 - p. 405 - 416.
hydrodynamica - vloeistoffen (liquids) - vloeistoffen (fluids) - stroming - poreus medium - kernmagnetische resonantie - kernmagnetische resonantiespectroscopie - hydrodynamics - liquids - fluids - flow - porous media - nuclear magnetic resonance - nuclear magnetic resonance spectroscopy
Displacement imaging is a recent, powerful NMR method with which distributions of displacements can be acquired of e.g. fluids within a porous medium. Both motion parallel and perpendicular to the flow direction may be observed within a time window of a few milliseconds to several seconds. By combining displacement imaging with the line scan technique, one-dimensionally resolved measurements with a high temporal resolution (<1 min) of the spatial dependency of motion can be obtained. Here we present displacement images of flow through two simple model systems for soil: an unconsolidated glass bead water system and a sintered glass bead filter. It is demonstrated that the combination of displacement imaging and spatial resolution along a line is important to access both bulk displacement and local displacements in relation to the local porosity.
NMR methods for imaging of transport processes in micro-porous systems.
As, H. van; Dusschoten, D. van - \ 1997
Geoderma 80 (1997). - ISSN 0016-7061 - p. 389 - 403.
hydrodynamica - vloeistoffen (liquids) - vloeistoffen (fluids) - stroming - poreus medium - kernmagnetische resonantie - kernmagnetische resonantiespectroscopie - hydrodynamics - liquids - fluids - flow - porous media - nuclear magnetic resonance - nuclear magnetic resonance spectroscopy
|NMR as a tool for easy routine analysis (in Dutch)
Eijck, P.C.M. van; Slaghek, T.E.A.M. ; Meer, P. van der - \ 1996
Voedingsmiddelentechnologie 29 (1996)21. - ISSN 0042-7934 - p. 54 - 55.
voedselindustrie - voedseltechnologie - kernmagnetische resonantie - kernmagnetische resonantiespectroscopie - food industry - food technology - nuclear magnetic resonance - nuclear magnetic resonance spectroscopy
Nuclear magnetic resonance methode
Probing water motion in heterogenous systems : a multi-parameter NMR approach
Dusschoten, D. van - \ 1996
Agricultural University. Promotor(en): T.J. Schaafsma; H. van As. - S.l. : Van Dusschoten - ISBN 9789054855286 - 141
vloeistofmechanica - water - beweging - stroming - kernmagnetische resonantie - kernmagnetische resonantiespectroscopie - fluid mechanics - water - movement - flow - nuclear magnetic resonance - nuclear magnetic resonance spectroscopy
In this Thesis a practical approach is presented to study water mobility in heterogeneous systems by a number of novel NMR sequences. The major part of this Thesis describes how the reliability of diffusion measurements can be improved using some of the novel NMR sequences. The reliability of the data can be further enhanced by combining different NMR characteristics in a single fit routine. In addition, a fast NMR sequence for flow measurements is shown. A wide variety of samples is used to demonstrate how the NMR sequences and the subsequent analysis work.
Throughout this Thesis the term heterogeneous system is used whenever a sample contains different physical or chemical environments or compositions, each of which influence the NMR signal in a distinguishable way. One of the effects of such a heterogeneous system is the variation of the magnetic susceptibility within the sample causing so-called in situ magnetic field gradients. These gradients lower the NMR signal amplitude and may cause a substantial deviation of the real diffusion constant from the one measured by NMR using pulsed field gradient spin echo or stimulated echo sequences. In the second half of Chapter 2 an improved version of the PFG multiple spin echo sequence is introduced which minimises the degrading effects of in situ field gradients. While using additional r.f. pulses between the pulsed field gradients to reach the desired reliability no compromises with regard to flexibility were necessary.
This flexibility is important to study the change of the apparent diffusion constant with the echo time. This phenomenon, superficially appearing as an artefact, arises because compartments, as for example a vacuole, not only have a characteristic diffusion constant but also a T 2 which may significantly differ from other T 2 's in the sample. In Chapter 3 it is shown how a T 2 difference can be used to separate diffusion constants (D) even if these have a similar magnitude. Using Diffusion Analysis by Relaxation Time Separation the diffusion constant for water in the vacuole, the cytoplasm and the extra cellular space, respectively, can be distinguished in apple parenchyma tissue. In the second half of Chapter 3 a fast implementation of DARTS PFG NMR is presented which is combined with NMR imaging. Here, spatially localised T 2 measurements are preceded by pulsed field gradients for diffusion weighting. In this way the diffusion constant and fractional amplitude of cerebral spinal fluid (CSF) and white and grey matter in cat brain can be measured. The validity of these measurements are supported by Monte Carlo simulations of computer generated 2D data sets.
Chapter 4 deals primarily with improving the DARTS technique without imaging (DARTS PFG MSE Carr Purcell Meiboom Gill) and determining the best resolution (for discriminating diffusion constants) which can be obtained, using Monte Carlo simulations on 2D data sets. The use of the 2D fitting routine is extensively studied. Furthermore, existing theoretical models are mutually compared and confronted with the results obtained by the PFG MSE CPMG sequence for different samples. Four heterogeneous samples with different complexity are studied, i.e.:
In Chapter 5 the subject of flow in heterogeneous systems is studied in further detail. In the first half of this Chapter novel flow measurements in and around the buccal cavity of a Carp are described. These measurements are performed on a standard medical imager without special, fast NMR sequences. The described data can therefore only be used in a qualitative manner. In the second half the line scan flow measurement is introduced. A temporal resolution of 16 ms can be obtained with this sequence allowing accurate, real time flow measurements. The combination of this line scan sequence with displacement imaging yields NMR images which picture the distribution of flow velocities over a line. Demonstrations of displacement imaging are performed in a tube with glass beads and in a pipe with a glass bead filter. The presented data can be used quantitatively.
The diffusion and flow measurements described in the Thesis all employ pulsed field gradients to encode for motion. Despite the obvious similarities between the measurements, the optimisation of the NMR sequences results in sequences which can be rather distinct. By careful tuning of the NMR sequences the range of displacements which can thus be measured lies between 5 μm and about 5 cm. This range and the fact that small differences in flow velocities and diffusion constants can be resolved, if necessary using other NMR characteristics, makes NMR a powerful tool to study water mobility in heterogeneous systems.