Metabolomic-Based Comparison of Daphnia magna and Japanese Medaka Responses After Exposure to Acetaminophen, Diclofenac, and Ibuprofen.

Pharmaceuticals are found in aquatic environments due to their widespread use and environmental persistence. To date, a range of impairments to aquatic organisms has been reported with exposure to pharmaceuticals; however, further comparisons of their impacts across different species on the molecular level are needed. In the present study, the crustacean Daphnia magna and the freshwater fish Japanese medaka, common model organisms in aquatic toxicity, were exposed for 48 h to the common analgesics acetaminophen (ACT), diclofenac (DCF), and ibuprofen (IBU) at sublethal concentrations. A targeted metabolomic-based approach, using liquid chromatography-tandem mass spectrometry to quantify polar metabolites from individual daphnids and fish was used. Multivariate analyses and metabolite changes identified differences in the metabolite profile for D. magna and medaka, with more metabolic perturbations for D. magna. Pathway analyses uncovered disruptions to pathways associated with protein synthesis and amino acid metabolism with D. magna exposure to all three analgesics. In contrast, medaka exposure resulted in disrupted pathways with DCF only and not ACT and IBU. Overall, the observed perturbations in the biochemistry of both organisms were different and consistent with assessments using other endpoints reporting that D. magna is more sensitive to pollutants than medaka in short-term studies. Our findings demonstrate that molecular-level responses to analgesic exposure can reflect observations of other endpoints, such as immobilization and mortality. Thus, environmental metabolomics can be a valuable tool for selecting sentinel species for the biomonitoring of freshwater ecosystems while also uncovering mechanistic information. Environ Toxicol Chem 2024;43:1339-1351. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Improved Photocatalytic Performance of TiO2-Nitrogen-Doped Graphene Quantum Dot Composites Mediated by Heterogeneous Interactions.

Photocatalysis is typically monitored via analysis of phases in isolation and focuses on the removal of a target analyte from the solution phase. Here we analyze the photocatalytic action of a TiO2-nitrogen-doped graphene quantum dot (NGQD) composite on a target analyte, phenol, using comprehensive multiphase NMR (CMP-NMR) which observes signals in solid, solution, and gel phases in situ. Phenol preferentially interacts with the composite photocatalyst compared to pure TiO2, increasing its effective concentration near the catalyst surface and its degradation rate. The presence of NGQDs in the composite reduced the fouling of the catalyst surface and caused a reduction of photogenerated intermediates. Increased heterogeneous interactions, likely mediated by π-π interactions, are hypothesized to cause each of these improvements in the observed photocatalytic performance by TiO2-NGQDs. CMP-NMR allows the elucidation of how the photocatalytic mechanism is enhanced via material design and provides a foundation for the development of efficient photocatalysts.

A simple 1H (12C/13C) filtered experiment to quantify and trace isotope enrichment in complex environmental and biological samples.

Nuclear magnetic resonance (NMR) based 13C tracing has broad applications across medical and environmental research. As many biological and environmental samples are heterogeneous, they experience considerable spectral overlap and relatively low signal. Here a 1D 1H-12C/13C is introduced that uses "in-phase/opposite-phase" encoding to simultaneously detect and discriminate both protons attached to 12C and 13C at full 1H sensitivity in every scan. Unlike traditional approaches that focus on the 12C/13C satellite ratios in a 1H spectrum, this approach creates separate sub-spectra for the 12C and 13C bound protons. These spectra can be used for both quantitative and qualitative analysis of complex samples with significant spectral overlap. Due to the presence of the 13C dipole, faster relaxation of the 1H-13C pairs results in slight underestimation compared to the 1H-12C pairs. However, this is easily compensated for, by collecting an additional reference spectrum, from which the absolute percentage of 13C can be calculated by difference. When combined with the result, 12C and 13C percent enrichment in both 1H-12C and 1H-13C fractions are obtained. As the approach uses isotope filtered 1H NMR for detection, it retains nearly the same sensitivity as a standard 1H spectrum. Here, a proof-of-concept is performed using simple mixtures of 12C and 13C glucose, followed by suspended algal cells with varying 12C /13C ratios representing a complex mixture. The results consistently return 12C/13C ratios that deviate less than 1 % on average from the expected. Finally, the sequence was used to monitor and quantify 13C% enrichment in Daphnia magna neonates which were fed a 13C diet over 1 week. The approach helped reveal how the organisms utilized the 12C lipids they are born with vs. the 13C lipids they assimilate from their diet during growth. Given the experiments simplicity, versatility, and sensitivity, we anticipate it should find broad application in a wide range of tracer studies, such as fluxomics, with applications spanning various disciplines.

Brewing alcohol 101: An undergraduate experiment utilizing benchtop NMR for quantification and process monitoring.

In recent years there has been a renewed interest in benchtop NMR. Given their lower cost of ownership, smaller footprint, and ease of use, they are especially suited as an educational tool. Here, a new experiment targeted at upper-year undergraduates and first-year graduate students follows the conversion of D-glucose into ethanol at low-field. First, high and low-field data on D-glucose are compared and students learn both the Hz and ppm scales and how J-coupling is field-independent. The students then acquire their own quantitative NMR datasets and perform the quantification using an Electronic Reference To Access In Vivo Concentration (ERETIC) technique. To our knowledge ERETIC is not currently taught at the undergraduate level, but has an advantage in that internal standards are not required; ideal for following processes or with future use in flow-based benchtop monitoring. Using this quantitative data, students can relate a simple chemical process (fermentation) back to more complex topics such as reaction kinetics, bridging the gaps between analytical and physical chemistry. When asked to reflect on the experiment, students had an overwhelmingly positive experience, citing agreement with learning objectives, ease of understanding the protocol, and enjoyment. Each of the respondents recommended this experiment as a learning tool for others. This experiment has been outlined for other instructors to utilize in their own courses across institutions, with the hope that a continued expansion of low-field NMR will increase accessibility and learning opportunities at the undergraduate level.

Slice through the water-Exploring the fundamental challenge of water suppression for benchtop NMR systems.

Benchtop NMR provides improved accessibility in terms of cost, space, and technical expertise. In turn, this encourages new users into the field of NMR spectroscopy. Unfortunately, many interesting samples in education and research, from beer to whole blood, contain significant amounts of water that require suppression in 1H NMR in order to recover sample information. However, due to the significant reduction in chemical shift dispersion in benchtop NMR systems, the sample signals are much closer to the water resonance compared to those in a corresponding high-field NMR spectrum. Therefore, simply translating solvent suppression experiments intended for high-field NMR instruments to benchtop NMR systems without careful consideration can be problematic. In this study, the effectiveness of several popular water suppression schemes was evaluated for benchtop NMR applications. Emphasis is placed on pulse sequences with no, or few, adjustable parameters making them easy to implement. These fall into two main categories: (1) those based on Pre-SAT including Pre-SAT, PURGE, NOESY-PR, and g-NOESY-PR and (2) those based on binomial inversion including JRS and W5-WATERGATE. Among these schemes, solvent suppression sequences based on Pre-SAT offer a general approach for easy solvent suppression for samples with higher analyte concentrations (sucrose standard and Redbull™). However, for human urine, binomial-like sequences were required. In summary, it is demonstrated that highly efficient water suppression approaches can be implemented on benchtop NMR systems in a simple manner, despite the limited spectral dispersion, further illustrating the potential for widespread implementation of these approaches in education and research.

Maternal exposure to polyethylene micro- and nanoplastics impairs umbilical blood flow but not fetal growth in pregnant mice.

While microplastics have been recently detected in human blood and the placenta, their impact on human health is not well understood. Using a mouse model of environmental exposure during pregnancy, our group has previously reported that exposure to polystyrene micro- and nanoplastics throughout gestation results in fetal growth restriction. While polystyrene is environmentally relevant, polyethylene is the most widely produced plastic and amongst the most commonly detected microplastic in drinking water and human blood. In this study, we investigated the effect of maternal exposure to polyethylene micro- and nanoplastics on fetal growth and placental function. Healthy, pregnant CD-1 dams were divided into three groups: 106 ng/L of 740-4990 nm polyethylene with surfactant in drinking water (n = 12), surfactant alone in drinking water (n = 12) or regular filtered drinking water (n = 11). At embryonic day 17.5, high-frequency ultrasound was used to investigate the placental and fetal hemodynamic responses following exposure. While maternal exposure to polyethylene did not impact fetal growth, there was a significant effect on placental function with a 43% increase in umbilical artery blood flow in the polyethylene group compared to controls (p < 0.01). These results suggest polyethylene has the potential to cause adverse pregnancy outcomes through abnormal placental function.

Polystyrene micro- and nanoplastics cause placental dysfunction in mice†.

Maternal exposure to microplastics and nanoplastics has been shown to result in fetal growth restriction in mice. In this study, we investigated the placental and fetal hemodynamic responses to plastics exposure in mice using high-frequency ultrasound. Healthy, pregnant CD-1 dams were given either 106 ng/L of 5 µm polystyrene microplastics or 106 ng/L of 50 nm polystyrene nanoplastics in drinking water throughout gestation and were compared with controls. Maternal exposure to both microplastics and nanoplastics resulted in evidence of placental dysfunction that was highly dependent on the particle size. The umbilical artery blood flow increased by 48% in the microplastic-exposed group and decreased by 25% in the nanoplastic-exposed group compared to controls (p < 0.05). The microplastic- and nanoplastic-exposed fetuses showed a significant decrease in the middle cerebral artery pulsatility index of 10% and 13%, respectively, compared to controls (p < 0.05), indicating vasodilation of the cerebral circulation, a fetal adaptation that is part of the brain sparing response to preserve oxygen delivery. Hemodynamic markers of placental dysfunction and fetal hypoxia were more pronounced in the group exposed to polystyrene nanoplastics, suggesting nanoplastic exposure during human pregnancy has the potential to disrupt fetal brain development, which in turn may cause suboptimal neurodevelopmental outcomes.

Metabolomics revealed disruptions in amino acid and antioxidant biochemistry in Daphnia magna exposed to industrial effluents associated with plastic and polymer production.

Industrial wastewater effluents are a major source of chemicals in aquatic environments, and many of these chemicals may negatively impact aquatic life. In this study, the crustacean Daphnia magna, a common model organism in ecotoxicity studies, was exposed for 48 h to nine different industrial effluent samples from manufacturing facilities associated with the production of plastics, polymers, and coating products at a range of dilutions: 10, 25, 50, 100% (undiluted). A targeted metabolomic-based approach using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to quantify polar metabolites from individual daphnids that survived the 48 h exposure. Multivariate analyses and metabolite changes revealed metabolic perturbations across all effluent samples studied, with non-monotonic responses and both up and downregulation relative to the unexposed control. Pathway analyses indicated the disruption of similar and distinct pathways, mostly connected to protein synthesis, amino acid metabolism, and antioxidant processes. Overall, we observed disruptions in Daphnia biochemistry that were similar across the effluent samples, but with unique features for each effluent sample. Additionally, non-monotonic heightened responses suggested additive and/or synergistic interactions between the chemicals within the industrial effluents. These findings demonstrate that targeted metabolomic approaches are a powerful tool for the biomonitoring of aquatic ecosystems in the context of complex mixtures, such as industrial wastewater effluents.

Low-field, not low quality: 1D simplification, selective detection, and heteronuclear 2D experiments for improving low-field NMR spectroscopy of environmental and biological samples.

Understanding environmental change is challenging and requires molecular-level tools to explain the physicochemical phenomena behind complex processes. Nuclear magnetic resonance (NMR) spectroscopy is a key tool that provides information on both molecular structures and interactions but is underutilized in environmental research because standard "high-field" NMR is financially and physically inaccessible for many and can be overwhelming to those outside of disciplines that routinely use NMR. "Low-field" NMR is an accessible alternative but has reduced sensitivity and increased spectral overlap, which is especially problematic for natural, heterogeneous samples. Therefore, the goal of this study is to investigate and apply innovative experiments that could minimize these challenges and improve low-field NMR analysis of environmental and biological samples. Spectral simplification (JRES, PSYCHE, singlet-only, multiple quantum filters), selective detection (GEMSTONE, DREAMTIME), and heteronuclear (reverse and CH3/CH2/CH-only HSQCs) NMR experiments are tested on samples of increasing complexity (amino acids, spruce resin, and intact water fleas) at-high field (500 MHz) and at low-field (80 MHz). A novel experiment called Doubly Selective HSQC is also introduced, wherein 1H signals are selectively detected based on the 1H and 13C chemical shifts of 1H-13C J-coupled pairs. The most promising approaches identified are the selective techniques (namely for monitoring), and the reverse and CH3-only HSQCs. Findings ultimately demonstrate that low-field NMR holds great potential for biological and environmental research. The multitude of NMR experiments available makes NMR tailorable to nearly any research need, and low-field NMR is therefore anticipated to become a valuable and widely used analytical tool moving forward.

HR-MAS DREAMTIME NMR for Slow Spinning ex Vivo and in Vivo Samples.

HR-MAS NMR is a powerful tool, capable of monitoring molecular changes in intact heterogeneous samples. However, one of the biggest limitations of 1H NMR is its narrow spectral width which leads to considerable overlap in complex natural samples. DREAMTIME NMR is a highly selective technique that allows users to isolate suites of metabolites from congested spectra. This permits targeted metabolomics by NMR and is ideal for monitoring specific processes. To date, DREAMTIME has only been employed in solution-state NMR, here it is adapted for HR-MAS applications. At high spinning speeds (>5 kHz), DREAMTIME works with minimal modifications. However, spinning over 3-4 kHz leads to cell lysis, and if maintaining sample integrity is necessary, slower spinning (<2.5 kHz) is required. Very slow spinning (≤500 Hz) is advantageous for in vivo analysis to increase organism survival; however, sidebands from water pose a problem. To address this, a version of DREAMTIME, termed DREAMTIME-SLOWMAS, is introduced. Both techniques are compared at 2500, 500, and 50 Hz, using ex vivo worm tissue. Following this, DREAMTIME-SLOWMAS is applied to monitor key metabolites of anoxic stress in living shrimp at 500 Hz. Thus, standard DREAMTIME works well under MAS conditions and is recommended for samples reswollen in D2O or spun >2500 Hz. For slow spinning in vivo or intact tissue samples, DREAMTIME-SLOWMAS provides an excellent way to target process-specific metabolites while maintaining sample integrity. Overall, DREAMTIME should find widespread application wherever targeted molecular information is required from complex samples with a high degree of spectral overlap.

Maternal exposure to polystyrene nanoplastics alters fetal brain metabolism in mice.

INTRODUCTION: Plastics used in everyday materials accumulate as waste in the environment and degrade over time. The impacts of the resulting particulate micro- and nanoplastics on human health remain largely unknown. In pregnant mice, we recently demonstrated that exposure to nanoplastics throughout gestation and during lactation resulted in changes in brain structure detected on MRI. One possible explanation for this abnormal postnatal brain development is altered fetal brain metabolism. OBJECTIVES: To determine the effect of maternal exposure to nanoplastics on fetal brain metabolism. METHODS: Healthy pregnant CD-1 mice were exposed to 50 nm polystyrene nanoplastics at a concentration of 106 ng/L through drinking water during gestation. Fetal brain samples were collected at embryonic day 17.5 (n = 18-21 per group per sex) and snap-frozen in liquid nitrogen. Magic angle spinning nuclear magnetic resonance was used to determine metabolite profiles and their relative concentrations in the fetal brain. RESULTS: The relative concentrations of gamma-aminobutyric acid (GABA), creatine and glucose were found to decrease by 40%, 21% and 30% respectively following maternal nanoplastic exposure when compared to the controls (p < 0.05). The change in relative concentration of asparagine with nanoplastic exposure was dependent on fetal sex (p < 0.005). CONCLUSION: Maternal exposure to polystyrene nanoplastics caused abnormal fetal brain metabolism in mice. The present study demonstrates the potential impacts of nanoplastic exposure during fetal development and motivates further studies to evaluate the risk to human pregnancies.

Identifying placental metabolic biomarkers of preterm birth using nuclear magnetic resonance of intact tissue samples.

INTRODUCTION: Our understanding of the etiology of preterm birth (PTB) is incomplete; however, recent evidence has found a strong association between placental dysfunction and PTB. Altered placental metabolism may precede placental dysfunction and therefore the study of placental metabolic profiles could identify early biomarkers of PTB. In this study, we evaluated the placental metabolome in PTB in intact tissue samples using nuclear magnetic resonance (NMR) and spectral editing. METHODS: Placental tissue samples were collected from nine term pregnancies and nine preterm pregnancies (<37 weeks' gestation). 1H NMR experiments on unprocessed tissue samples were performed using a high field magnet (500 MHz spectrometer) and a comprehensive multiphase NMR probe. The relative concentrations of 23 metabolites were corrected for gestational age and compared between groups. RESULTS: The relative concentration of valine, glutamate and creatine were significantly decreased while alanine, choline and glucose were elevated in placentas from PTB pregnancies compared to controls (p < 0.05). Multivariate analysis using principal component analysis showed the PTB and control groups were significantly separated (p < 0.0001) and pathway analysis identified perturbations in the glycine, serine and threonine metabolism, aminoacyl-tRNA biosynthesis and valine, leucine and isoleucine biosynthesis pathways. CONCLUSION: PTB is associated with significant alterations in placental metabolism. This study helps improve our understanding of the etiology of PTB. It also highlights the potential for small molecule metabolites to serve as placental metabolic biomarkers to aid in the prediction and diagnosis of PTB. The results can be translated to clinical use via in utero magnetic resonance spectroscopy.

Development of Low-Magnetic Susceptibility Microcoils via 5-Axis Machining for Analysis of Biological and Environmental Samples.

In environmental research, it is critical to understand how toxins impact invertebrate eggs and egg banks, which, due to their tiny size, are very challenging to study by conventional nuclear magnetic resonance (NMR) spectroscopy. Microcoil technology has been extensively utilized to enhance the mass-sensitivity of NMR. In a previous study, 5-axis computer numerical control (CNC) micromilling (shown to be a viable alternative to traditional microcoil production methods) was used to create a prototype copper slotted-tube resonator (STR). Despite the excellent limit of detection (LOD) of the resonator, the quality of the line shape was very poor due to the magnetic susceptibility of the copper resonator itself. This is best solved using magnetic susceptibility-matched materials. In this study, approaches are investigated that improve the susceptibility while retaining the versatility of coil milling. One method involves machining STRs from various copper/aluminum alloys, while the other involves machining ones from an aluminum 2011 alloy and electroplating them with copper. In all cases, combining copper and aluminum to produce resonators resulted in improved line shape and SNR compared to pure copper resonators due to their reduced magnetic susceptibility. However, the copper-plated aluminum resonators showed optimal performance from the devices tested. The enhanced LOD of these STRs allowed for the first 1H-13C heteronuclear multiple quantum coherence (HMQC) of a single intact 13C-labeled Daphnia magna egg (∼4 µg total biomass). This is a key step toward future screening programs that aim to elucidate the toxic processes in aquatic eggs.

Cutting without a Knife: A Slice-Selective 2D 1H-13C HSQC NMR Sequence for the Analysis of Inhomogeneous Samples.

Nuclear magnetic resonance (NMR) is a powerful technique with applications ranging from small molecule structure elucidation to metabolomics studies of living organisms. Typically, solution-state NMR requires a homogeneous liquid, and the whole sample is analyzed as a single entity. While adequate for homogeneous samples, such an approach is limited if the composition varies as would be the case in samples that are naturally heterogeneous or layered. In complex samples such as living organisms, magnetic susceptibility distortions lead to broad 1H line shapes, and thus, the additional spectral dispersion afforded by 2D heteronuclear experiments is often required for metabolite discrimination. Here, a novel, slice-selective 2D, 1H-13C heteronuclear single quantum coherence (HSQC) sequence was developed that exclusively employs shaped pulses such that only spins in the desired volume are perturbed. In turn, this permits multiple volumes in the tube to be studied during a single relaxation delay, increasing sensitivity and throughput. The approach is first demonstrated on standards and then used to isolate specific sample/sensor elements from a microcoil array and finally study slices within a living earthworm, allowing metabolite changes to be discerned with feeding. Overall, slice-selective NMR is demonstrated to have significant potential for the study of layered and other inhomogeneous samples of varying complexity. In particular, its ability to select subelements is an important step toward developing microcoil receive-only arrays to study environmental toxicity in tiny eggs, cells, and neonates, whereas localization in larger living species could help better correlate toxin-induced biochemical responses to the physical localities or organs involved.

Application of 15N-Edited 1H-13C Correlation NMR Spectroscopy─Toward Fragment-Based Metabolite Identification and Screening via HCN Constructs.

Many key building blocks of life contain nitrogen moieties. Despite the prevalence of nitrogen-containing metabolites in nature, 15N nuclei are seldom used in NMR-based metabolite assignment due to their low natural abundance and lack of comprehensive chemical shift databases. However, with advancements in isotope labeling strategies, 13C and 15N enriched metabolites are becoming more common in metabolomic studies. Simple multidimensional nuclear magnetic resonance (NMR) experiments that correlate 1H and 15N via single bond 1JNH or multiple bond 2-3JNH couplings using heteronuclear single quantum coherence (HSQC) or heteronuclear multiple bond coherence are well established and routinely applied for structure elucidation. However, a 1H-15N correlation spectrum of a metabolite mixture can be difficult to deconvolute, due to the lack of a 15N specific database. In order to bridge this gap, we present here a broadband 15N-edited 1H-13C HSQC NMR experiment that targets metabolites containing 15N moieties. Through this approach, nitrogen-containing metabolites, such as amino acids, nucleotide bases, and nucleosides, are identified based on their 13C, 1H, and 15N chemical shift information. This approach was tested and validated using a [15N, 13C] enriched Daphnia magna (water flea) metabolite extract, where the number of clearly resolved 15N-containing peaks increased from only 11 in a standard HSQC to 51 in the 15N-edited HSQC, and the number of obscured peaks decreased from 59 to just 7. The approach complements the current repertoire of NMR techniques for mixture deconvolution and holds considerable potential for targeted metabolite NMR in 15N, 13C enriched systems.

Exploring the Potential of Broadband Complementary Metal Oxide Semiconductor Micro-Coil Nuclear Magnetic Resonance for Environmental Research.

With sensitivity being the Achilles' heel of nuclear magnetic resonance (NMR), the superior mass sensitivity offered by micro-coils can be an excellent choice for tiny, mass limited samples such as eggs and small organisms. Recently, complementary metal oxide semiconductor (CMOS)-based micro-coil transceivers have been reported and demonstrate excellent mass sensitivity. However, the ability of broadband CMOS micro-coils to study heteronuclei has yet to be investigated, and here their potential is explored within the lens of environmental research. Eleven nuclei including 7Li, 19F, 31P and, 205Tl were studied and detection limits in the low to mid picomole range were found for an extended experiment. Further, two environmentally relevant samples (a sprouting broccoli seed and a D. magna egg) were successfully studied using the CMOS micro-coil system. 13C NMR was used to help resolve broad signals in the 1H spectrum of the 13C enriched broccoli seed, and steady state free precession was used to improve the signal-to-noise ratio by a factor of six. 19F NMR was used to track fluorinated contaminants in a single D. magna egg, showing potential for studying egg-pollutant interactions. Overall, CMOS micro-coil NMR demonstrates significant promise in environmental research, especially when the future potential to scale to multiple coil arrays (greatly improving throughput) is considered.

Exploring Proton-Only NMR Experiments and Filters for Daphnia In Vivo: Potential and Limitations.

Environmental metabolomics provides insight into how anthropogenic activities have an impact on the health of an organism at the molecular level. Within this field, in vivo NMR stands out as a powerful tool for monitoring real-time changes in an organism's metabolome. Typically, these studies use 2D 13C-1H experiments on 13C-enriched organisms. Daphnia are the most studied species, given their widespread use in toxicity testing. However, with COVID-19 and other geopolitical factors, the cost of isotope enrichment increased ~6-7 fold over the last two years, making 13C-enriched cultures difficult to maintain. Thus, it is essential to revisit proton-only in vivo NMR and ask, "Can any metabolic information be obtained from Daphnia using proton-only experiments?". Two samples are considered here: living and whole reswollen organisms. A range of filters are tested, including relaxation, lipid suppression, multiple-quantum, J-coupling suppression, 2D 1H-1H experiments, selective experiments, and those exploiting intermolecular single-quantum coherence. While most filters improve the ex vivo spectra, only the most complex filters succeed in vivo. If non-enriched organisms must be used, then, DREAMTIME is recommended for targeted monitoring, while IP-iSQC was the only experiment that allowed non-targeted metabolite identification in vivo. This paper is critically important as it documents not just the experiments that succeed in vivo but also those that fail and demonstrates first-hand the difficulties associated with proton-only in vivo NMR.

Targeted Compound Selection with Increased Sensitivity in 13C-Enriched Biological and Environmental Samples Using 13C-DREAMTIME in Both High-Field and Low-Field NMR.

Chemical characterization of complex mixtures by Nuclear Magnetic Resonance (NMR) spectroscopy is challenging due to a high degree of spectral overlap and inherently low sensitivity. Therefore, NMR experiments that reduce overlap and increase signal intensity hold immense potential for the analysis of mixtures such as biological and environmental media. Here, we introduce a 13C version of DREAMTIME (Designed Refocused Excitation And Mixing for Targets In Vivo and Mixture Elucidation) NMR, which, when analyzing 13C-enriched materials, allows the user to selectively detect only the compound(s) of interest and remove all other peaks in a 13C spectrum. Selected peaks can additionally be "focused" into sharp "spikes" to increase sensitivity. 13C-DREAMTIME is first demonstrated at high field strength (500 MHz) with simultaneous selection of eight amino acids in a 13C-enriched cell free amino acid mixture and of six metabolites in an extract of 13C-enriched green algae and demonstrated at low field strength (80 MHz) with a standard solution of 13C-d-glucose and 13C-l-phenylalanine. 13C-DREAMTIME is then applied at high-field to analyze metabolic changes in 13C-enrichedDaphnia magna after exposure to polystyrene "microplastics," as well as at low-field to track fermentation of 13C-d-glucose using wine yeast. Ultimately, 13C-DREAMTIME reduces spectral overlap as only selected compounds are recorded, resulting in the detection of analyte peaks that may otherwise not have been discernable. In combination with focusing, up to a 6-fold increase in signal intensity can be obtained for a given peak. 13C-DREAMTIME is a promising experiment type for future reaction monitoring and for tracking metabolic processes with 13C-enriched compounds.

Compositional changes in the humin fraction resulting from the long-term cultivation of an Irish grassland soil: Evidence from FTIR and multi-NMR spectroscopies.

Soil humin (HN), a major long-term sink for carbon in the pedosphere, plays a key role in the global carbon cycle, and has been less extensively studied than the humic and fulvic acids components. There are increasing concerns about the depletions of soil organic matter (SOM) arising from modern soil cultivation practices but there has been little focus on how HN can be altered as the result. This study has compared the HN components in a soil under cultivation for wheat for >30 years with those from an adjacent contiguous soil that had been under long-term grass for all that time. A urea-fortified basic solution isolated additional humic fractions from soils that had been exhaustively extracted in basic media. Then further exhaustive extractions of the residual soil material with dimethyl sulfoxide, amended with sulphuric acid isolated what may be called the "true" HN fraction. The long-term cultivation resulted in a loss of 53 % soil organic carbon in the surface soil. Infrared and multi-NMR spectroscopies showed the "true" HN to be dominated by aliphatic hydrocarbons and carboxylated structures, but with clear evidence for lesser amounts of carbohydrate and peptide materials, and with weaker evidence for lignin-derived substances. These lesser-amount structures can be sorbed on the soil mineral colloid surfaces and/or covered by the hydrophobic HN component or entrained within these which have strong affinities for the mineral colloids. HN from the cultivated site contained less carbohydrate and more carboxyl groups suggesting slow transformations took place resulting from the cultivation, but these were much slower than for the other components of SOM. It is recommended that a study be made of the HN in a soil under long-term cultivation for which the SOM content has reached a steady state and where HN will be expected to dominate the components of SOM.