RÉSUMÉ
In-vivo Nuclear Magnetic Resonance (NMR) spectroscopy is a unique and powerful approach for understanding sublethal toxicity, recovery, and elucidating a contaminant's toxic mode of action. However, magnetic susceptibility distortions caused by the organisms, along with sample complexity, lead to broad and overlapping 1D NMR spectra. As such, 2D NMR in combination with 13C enrichment (to increase signal) is a requirement for metabolite assignment and monitoring using high field in-vivo flow based NMR. Despite this, it is not clear which NMR experiment and probe combinations are the most appropriate for such studies. In terms of experiments, 1H-13C Heteronuclear Single Quantum Coherence (HSQC) and 13C-1H Heteronuclear Correlation Spectroscopy (HETCOR) experiments are logical choices for molecular fingerprinting. HSQC uses 1H for detection and thus will be the most sensitive, while HETCOR uses 13C for detection, which benefits from improved spectral dispersion (i.e. a larger chemical shift range) and avoids detection of the huge in-vivo water signal which can be problematic in HSQC. NMR probes are available in two variations, inverse (inner coil 1H) which is best suited to 1H detection and observe (inner coil 13C) which is ideal for 13C detection. To further complicate matters, the low biomass in many aquatic organisms makes cryoprobes desirable, however, changing cryoprobes is time prohibitive, requiring at least a day to warmup and cool down, meaning only a single probe can be used to monitor "real-time" in-vivo responses. The key questions become: Is it best to use HSQC on an inverse cryoprobe and accept a compromised HETCOR? Or is it best to use HETCOR on an observe cryoprobe and accept a compromised HSQC? Here these questions are explored using living 13C enriched Daphnia as the test case. The number of metabolites identified across the different probe/experiment combinations are compared over a range of experiment times. Finally, the probes/experiments are compared to monitor an anoxic stress response. Both probes and experiments prove to be quite robust, albeit HSQC identified slightly more metabolites in most cases. HETCOR did nearly as-well and because of the lack of water complications would be the most accessible approach for researchers to apply in-vivo NMR to 13C enriched organisms, both in terms of experimental setup and flow system design. This said, when using an optimized flow system, HSQC did identify the most metabolites and an inverse probe design offers the most potential for 1H-only approaches which are continuously being developed and have the potential to eventually overcome the current limitation that requires 13C enriched organisms.
Sujet(s)
Imagerie par résonance magnétique , Métabolomique , Animaux , Daphnia , Spectroscopie par résonance magnétique , EauRÉSUMÉ
In vivo nuclear magnetic resonance (NMR) is a powerful analytical tool for probing complex biological processes inside living organisms. However, due to magnetic susceptibility broadening, which produces broad lines in one-dimensional NMR, 1H-13C two-dimensional (2D) NMR is required for metabolite monitoring in vivo. As each 2D experiment is time-consuming, often hours, this limits the temporal resolution over which in vivo processes can be monitored. Furthermore, to understand concentration-dependent responses, studies are traditionally repeated using different contaminant and toxin concentrations, which can make studies prohibitively long (potentially months). In this study, time-resolved non-uniform sampling NMR is performed in the presence of a contaminant concentration sweep. The result is that the lowest concentration that elicits a metabolic response can be rapidly detected, while the metabolic pathways impacted provide information about the toxic mode of action of the toxin. The lowest concentration of bisphenol A (BPA) that induces a response was â¼0.1 mg/L (detected in just 16 min), while changes in different metabolites suggest a complex multipathway response that leads to protein degradation at higher BPA concentrations. This proof of concept shows it is possible, on the basis of "real-time" organism responses, to identify the sublethal concentration at which a toxin impacts an organism and thus represents an essential analytical tool for the next generation of toxicity-based research and monitoring.
Sujet(s)
Composés benzhydryliques/toxicité , Daphnia/effets des médicaments et des substances chimiques , Decapoda (crustacea)/effets des médicaments et des substances chimiques , Imagerie par résonance magnétique/méthodes , Phénols/toxicité , Animaux , Composés benzhydryliques/administration et posologie , Relation dose-effet des médicaments , Oestrogènes nonstéroïdiens/administration et posologie , Oestrogènes nonstéroïdiens/toxicité , Phénols/administration et posologieRÉSUMÉ
In vivo nuclear magnetic resonance (NMR) is rapidly evolving as a critical tool as it offers real-time metabolic information, which is crucial for delineating complex toxic response pathways in living systems. Organisms such as Daphnia magna (water fleas) and Hyalella azteca (freshwater shrimps) are commonly 13C-enriched to increase the signal in NMR experiments. A key goal of in vivo NMR is to monitor how molecules (nutrients, contaminants, or drugs) are metabolized. Conventionally, these studies would normally involve using a 13C-enriched probe molecule and feeding this to an organism at natural abundance, in turn allowing the fate of the probe molecule to be selectively analyzed. The drawback of such an approach is that there is a limited range of 13C-enriched probe molecules, and if available, they are extremely cost prohibitive. Uniquely, when utilizing 13C organisms, a reverse strategy of isotopic filtering becomes possible. The concept described here uses 1H detection in combination with a 13C filter on living organisms. The purpose is to suppress all 1H signals from the organism (i.e., 1H attached to 13C), leaving only the probe molecule (1H attached to 12C). Because the probe molecule can be selectively observed using this approach, it then makes it possible to follow and discern processes such as bioconversion, bioaccumulation, and excretion in vivo. As the approach uses 1H detection, it provides excellent detection limits in the nanogram range. In this article, the approach is introduced, optimized on standards, and then applied to follow nicotine biotransformation and lipid assimilation in vivo to demonstrate the concept.
Sujet(s)
Spectroscopie par résonance magnétique/méthodes , Métabolomique/méthodes , Animaux , Biotransformation , Spectroscopie par résonance magnétique du carbone-13/méthodes , Daphnia/métabolisme , Decapoda (crustacea)/métabolisme , Mobilisation des lipides , Nicotine/pharmacocinétique , Spectroscopie par résonance magnétique du proton/méthodesRÉSUMÉ
In vivo NMR of small 13C-enriched aquatic organisms is developing as a powerful tool to detect and explain toxic stress at the biochemical level. Amino acids are a very important category of metabolites for stress detection as they are involved in the vast majority of stress response pathways. As such, they are a useful proxy for stress detection in general, which could then be a trigger for more in-depth analysis of the metabolome. 1H-13C heteronuclear single quantum coherence (HSQC) is commonly used to provide additional spectral dispersion in vivo and permit metabolite assignment. While some amino acids can be assigned from HSQC, spectral overlap makes monitoring them in vivo challenging. Here, an experiment typically used to study protein structures is adapted for the selective detection of amino acids inside living Daphnia magna (water fleas). All 20 common amino acids can be selectively detected in both extracts and in vivo. By monitoring bisphenol-A exposure, the in vivo amino acid-only approach identified larger fluxes in a greater number of amino acids when compared to published works using extracts from whole organism homogenates. This suggests that amino acid-only NMR of living organisms may be a very sensitive tool in the detection of stress in vivo and is highly complementary to more traditional metabolomics-based methods. The ability of selective NMR experiments to help researchers to "look inside" living organisms and only detect specific molecules of interest is quite profound and paves the way for the future development of additional targeted experiments for in vivo research and monitoring.
RÉSUMÉ
Current research is attempting to address more complex questions than ever before. As such, the need to follow complex processes in intact media and mixtures is becoming commonplace. Here, a targeted NMR experiment is introduced which selectively detects the formation of 13C-12C bonds in mixtures. This study introduces the experiment on simple standards, and then demonstrates the potential on increasingly complex processes including: fermentation, Arabidopsis thaliana germination/early growth, and metabolism in Daphnia magna both ex vivo and in vivo. As signals from the intact 12C and 13C pools are themselves filtered out, correlations are only observed when a component from each pool combines (i.e. new 13C-12C bonds) in the formation of new structures. This targeted approach significantly reduces the complexity of the mixtures and provides information on the fate and reactivity of carbon in environmental and biological processes. The experiment has application to follow bond formation wherever two pools of carbon are brought together, be it the incorporation of 13C enriched food into a living organism's biomass, or the degradation of 13C enriched plant material in soil.
Sujet(s)
Carbone/composition chimique , Mélanges complexes/analyse , Animaux , Arabidopsis/composition chimique , Arabidopsis/croissance et développement , Arabidopsis/métabolisme , Isotopes du carbone/composition chimique , Mélanges complexes/métabolisme , Daphnia/métabolisme , Fermentation , Spectroscopie par résonance magnétiqueRÉSUMÉ
Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for the non-targeted metabolomics of intact biofluids and even living organisms. However, spectral overlap can limit the information that can be obtained from 1D 1H NMR. For example, magnetic susceptibility broadening in living organisms prevents any metabolic information being extracted from solution-state 1D 1H NMR. Conversely, the additional spectral dispersion afforded by 2D 1H-13C NMR allows a wide range of metabolites to be assigned in-vivo in 13C enriched organisms, as well as a greater depth of information for biofluids in general. As such, 2D 1H-13C NMR is becoming more and more popular for routine metabolic screening of very complex samples. Despite this, there are only a very limited number of statistical software packages that can handle 2D NMR datasets for chemometric analysis. In comparison, a wide range of commercial and free tools are available for analysis of 1D NMR datasets. Overtime, it is likely more software solutions will evolve that can handle 2D NMR directly. In the meantime, this application note offers a simple alternative solution that converts 2D 1H-13C Heteronuclear Single Quantum Correlation (HSQC) data into a 1D "spikelet" format that preserves not only the 2D spectral information, but also the 2D dispersion. The approach allows 2D NMR data to be converted into a standard 1D Bruker format that can be read by software packages that can only handle 1D NMR data. This application note uses data from Daphnia magna (water fleas) in-vivo to demonstrate how to generate and interpret the converted 1D spikelet data from 2D datasets, including the code to perform the conversion on Bruker spectrometers.
RÉSUMÉ
In vivo nuclear magnetic resonance (NMR) spectroscopy is a particularly powerful technique, since it allows samples to be analyzed in their natural, unaltered state, criteria paramount for living organisms. In this study, a novel continuous low-volume flow system, suitable for in vivo NMR metabolomics studies, is demonstrated. The system allows improved locking, shimming, and water suppression, as well as allowing the use of trace amounts of expensive toxic contaminants or low volumes of precious natural environmental samples as stressors. The use of a double pump design with a sump slurry pump return allows algal food suspensions to be continually supplied without the need for filters, eliminating the possibility of clogging and leaks. Using the flow system, the living organism can be kept alive without stress indefinitely. To evaluate the feasibility and applicability of the flow system, changes in the metabolite profile of 13C enriched Daphnia magna over a 24-h period are compared when feeding laboratory food vs exposing them to a natural algal bloom sample. Clear metabolic changes are observed over a range of metabolites including carbohydrates, lipids, amino acids, and a nucleotide demonstrating in vivo NMR as a powerful tool to monitor environmental stress. The particular bloom used here was low in microcystins, and the metabolic stress impacts are consistent with the bloom being a poor food source forcing the Daphnia to utilize their own energy reserves.
Sujet(s)
Spectroscopie par résonance magnétique/méthodes , Chlamydomonas reinhardtii/composition chimique , Chlamydomonas reinhardtii/métabolisme , Conception d'appareillage , Spectroscopie par résonance magnétique/instrumentation , Analyse multifactorielle , Oxygène/composition chimique , Solutions , Eau/composition chimiqueRÉSUMÉ
Part review, part perspective, this article examines the applications and potential of in-vivo Nuclear Magnetic Resonance (NMR) for understanding environmental toxicity. In-vivo NMR can be applied in high field NMR spectrometers using either magic angle spinning based approaches, or flow systems. Solution-state NMR in combination with a flow system provides a low stress approach to monitor dissolved metabolites, while magic angle spinning NMR allows the detection of all components (solutions, gels and solids), albeit with additional stress caused by the rapid sample spinning. With in-vivo NMR it is possible to use the same organisms for control and exposure studies (controls are the same organisms prior to exposure inside the NMR). As such individual variability can be reduced while continual data collection over time provides the temporal resolution required to discern complex interconnected response pathways. When multidimensional NMR is combined with isotopic labelling, a wide range of metabolites can be identified in-vivo providing a unique window into the living metabolome that is highly complementary to more traditional metabolomics studies employing extracts, tissues, or biofluids.