Evaluation of double-tuned single-sided planar microcoils for the analysis of small 13 C enriched biological samples using 1 H-13 C 2D heteronuclear correlation NMR spectroscopy.

Microcoils provide a cost-effective approach to improve detection limits for mass-limited samples. Single-sided planar microcoils are advantageous in comparison to volume coils, in that the sample can simply be placed on top. However, the considerable drawback is that the RF field that is produced by the coil decreases with distance from the coil surface, which potentially limits more complex multi-pulse NMR pulse sequences. Unfortunately, 1 H NMR alone is not very informative for intact biological samples due to line broadening caused by magnetic susceptibility distortions, and 1 H-13 C 2D NMR correlations are required to provide the additional spectral dispersion for metabolic assignments in vivo or in situ. To our knowledge, double-tuned single-sided microcoils have not been applied for the 2D 1 H-13 C analysis of intact 13 C enriched biological samples. Questions include the following: Can 1 H-13 C 2D NMR be performed on single-sided planar microcoils? If so, do they still hold sensitivity advantages over conventional 5 mm NMR technology for mass limited samples? Here, 2D 1 H-13 C HSQC, HMQC, and HETCOR variants were compared and then applied to 13 C enriched broccoli seeds and Daphnia magna (water fleas). Compared to 5 mm NMR probes, the microcoils showed a sixfold improvement in mass sensitivity (albeit only for a small localized region) and allowed for the identification of metabolites in a single intact D. magna for the first time. Single-sided planar microcoils show practical benefit for 1 H-13 C NMR of intact biological samples, if localized information within ~0.7 mm of the 1 mm I.D. planar microcoil surface is of specific interest.

Selective Amino Acid-Only in Vivo NMR: A Powerful Tool To Follow Stress Processes.

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.

1D "Spikelet" Projections from Heteronuclear 2D NMR Data-Permitting 1D Chemometrics While Preserving 2D Dispersion.

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.

Assessing the potential of quantitative 2D HSQC NMR in 13C enriched living organisms.

In vivo Nuclear Magnetic Resonance (NMR) spectroscopy has great potential to interpret the biochemical response of organisms to their environment, thus making it an essential tool in understanding toxic mechanisms. However, magnetic susceptibility distortions lead to 1D NMR spectra of living organisms with lines that are too broad to identify and quantify metabolites, necessitating the use of 2D 1H-13C Heteronuclear Single Quantum Coherence (HSQC) as a primary tool. While quantitative 2D HSQC is well established, to our knowledge it has yet to be applied in vivo. This study represents a simple pilot study that compares two of the most popular quantitative 2D HSQC approaches to determine if quantitative results can be directly obtained in vivo in isotopically enriched Daphnia magna (water flea). The results show the perfect-HSQC experiment performs very well in vivo, but the decoupling scheme used is critical for accurate quantitation. An improved decoupling approach derived using optimal control theory is presented here that improves the accuracy of metabolite concentrations that can be extracted in vivo down to micromolar concentrations. When combined with 2D Electronic Reference To access In vivo Concentrations (ERETIC) protocols, the protocol allows for the direct extraction of in vivo metabolite concentrations without the use of internal standards that can be detrimental to living organisms. Extracting absolute metabolic concentrations in vivo is an important first step and should, for example, be important for the parameterization as well as the validation of metabolic flux models in the future.

Improvements in lipid suppression for 1 H NMR-based metabolomics: Applications to solution-state and HR-MAS NMR in natural and in vivo samples.

Proton nuclear magnetic resonance (NMR) spectra of intact biological samples often show strong contributions from lipids, which overlap with signals of interest from small metabolites. Pioneering work by Diserens et al. demonstrated that the relative differences in diffusivity and relaxation of lipids versus small metabolites could be exploited to suppress lipid signals, in high-resolution magic angle spinning (HR-MAS) NMR spectroscopy. In solution-state NMR, suspended samples can exhibit very broad water signals, which are challenging to suppress. Here, improved water suppression is incorporated into the sequence, and the Carr-Purcell-Meiboom-Gill sequence (CPMG) train is replaced with a low-power adiabatic spinlock that reduces heating and spectral artefacts seen with longer CPMG filters. The result is a robust sequence that works well in both HR-MAS as well as static solution-state samples. Applications are also extended to include in vivo organisms. For solution-state NMR, samples containing significant amount of fats such as milk and hemp hearts seeds are used to demonstrate the technique. For HR-MAS, living earthworms (Eisenia fetida) and freshwater shrimp (Hyalella azteca) are used for in vivo applications. Lipid suppression techniques are essential for non-invasive NMR-based analysis of biological samples with a high-lipid content and adds to the suite of experiments advantageous for in vivo environmental metabolomics.

Development and Application of a Low-Volume Flow System for Solution-State in Vivo NMR.

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.

Comprehensive Multiphase (CMP) NMR Monitoring of the Structural Changes and Molecular Flux Within a Growing Seed.

A relatively recent technique termed comprehensive multiphase (CMP) NMR spectroscopy was used to investigate the growth and associated metabolomic changes of 13C-labeled wheat seeds and germinated seedlings. CMP-NMR enables the study of all phases in intact samples (i.e., liquid, gel-like, semisolid, and solid), by combining all required electronics into a single NMR probe, and can be used for investigating biological processes such as seed germination. All components, from the most liquid-like (i.e., dissolved metabolites) to the most rigid or solid-like (seed coat) were monitored in situ over 4 days. A wide range of metabolites were identified, and after 96 h of germination, the number of metabolites in the mobile phase more than doubled in comparison to 0 h (dry seed). This work represents the first application of CMP-NMR to follow biological processes in plants.

Analysis of DOM phototransformation using a looped NMR system integrated with a sunlight simulator.

Photochemical transformation plays an important role in functionalizing and degrading dissolved organic matter (DOM), producing one of the most complex mixtures known. In this study, using a flow-based design, nuclear magnetic resonance (NMR) spectroscopy is directly interfaced with a sunlight simulator enabling the study of DOM photodegradation in situ with high temporal resolution over 5 days. Samples from Suwannee River (Florida), Nordic Reservoir (Norway), and Pony Lake (Antarctic) are studied. Phototransformation of DOM is dominated by the degradation of aromatics and unsaturated structures (many arising from lignin) into carboxylated and hydroxylated products. To assess longer term changes, the samples were continuously irradiated for 17.5 days, followed by the identification a wide range of compounds and assessment of their fate using off-line 2D-NMR. This study demonstrates the applicability of the looped system to follow degradation in a non-targeted fashion (the mixture as a whole) and target analysis (tracing specific metabolites), which holds great potential to study the fate and transformation of contaminants and nutrients in the presence of DOM. It also demonstrates that components that remain unresolved in 1D NMR can be identified using 2D methods.

Development of an in Situ NMR Photoreactor To Study Environmental Photochemistry.

Photochemistry is a key environmental process directly linked to the fate, source, and toxicity of pollutants in the environment. This study explores two approaches for integrating light sources with nuclear magnetic resonance (NMR) spectroscopy: sample irradiation using a "sunlight simulator" outside the magnet versus direct irradiation of the sample inside the magnet. To assess their applicability, the in situ NMR photoreactors were applied to a series of environmental systems: an atmospheric pollutant (p-nitrophenol), crude oil extracts, and groundwater. The study successfully illustrates that environmentally relevant aqueous photochemical processes can be monitored in situ and in real time using NMR spectroscopy. A range of intermediates and degradation products were identified and matched to the literature. Preliminary measurements of half-lives were also obtained from kinetic curves. The sunlight simulator was shown to be the most suitable model to explore environmental photolytic processes in situ. Other light sources with more intense UV output hold potential for evaluating UV as a remediation alternative in areas such as wastewater treatment plants or oil spills. Finally, the ability to analyze the photolytic fate of trace chemicals at natural abundance in groundwater, using a cryogenic probe, demonstrates the viability of NMR spectroscopy as a powerful and complementary technique for environmental applications in general.

Identification of aquatically available carbon from algae through solution-state NMR of whole (13)C-labelled cells.

Green algae and cyanobacteria are primary producers with profound impact on food web functioning. Both represent key carbon sources and sinks in the aquatic environment, helping modulate the dissolved organic matter balance and representing a potential biofuel source. Underlying the impact of algae and cyanobacteria on an ecosystem level is their molecular composition. Herein, intact (13)C-labelled whole cell suspensions of Chlamydomonas reinhardtii, Chlorella vulgaris and Synechocystis were studied using a variety of 1D and 2D (1)H/(13)C solution-state nuclear magnetic resonance (NMR) spectroscopic experiments. Solution-state NMR spectroscopy of whole cell suspensions is particularly relevant as it identifies species that are mobile (dissolved or dynamic gels), 'aquatically available' and directly contribute to the aquatic carbon pool upon lysis, death or become a readily available food source on consumption. In this study, a wide range of metabolites and structural components were identified within the whole cell suspensions. In addition, significant differences in the lipid/triacylglyceride (TAG) content of green algae and cyanobacteria were confirmed. Mobile species in algae are quite different from those in abundance in 'classic' dissolved organic matter (DOM) indicating that if algae are major contributors to DOM, considerable selective preservation of minor components (e.g. sterols) or biotransformation would have to occur. Identifying the metabolites and dissolved components within algal cells by NMR permits future studies of carbon transfer between species and through the food chain, whilst providing a foundation to better understand the role of algae in the formation of DOM and the sequestration/transformation of carbon in aquatic environments.