André J. Simpson

HILIC-NMR: toward the identification of individual molecular components in dissolved organic matter.

This article presents research targeted toward the isolation and detection of unique molecular structures from what is believed to be the worlds most complex organic mixture: dissolved organic matter (DOM). Hydrophilic interaction chromatography (HILIC) was used to separate Suwannee River DOM (SRDOM) into 80 fractions, simplified to the extent that detection with nuclear magnetic resonance spectroscopy (NMR) results in many sharp signals that are indicative of individual compounds, some of which are identifiable with multidimensional NMR. Parallel factor analysis (PARAFAC) of fluorescence excitation-emission matrices (EEMs) was additionally employed on HILIC-simplified fractions to further confirm the effectiveness of the HILIC separations as well as draw insight into how structural characteristics relate to DOM fluorescence signals. Findings suggest that material believed to be derived from both cyclic and linear terpenoids was dominant in the most hydrophobic fractions as were the majority of the fluorescence signals, whereas hydrophilic material was highly correlated with carbohydrate-type structures as well as high contributions from amino acid fluorescence. NMR spectra of DOM, typically featureless mounds, are substantially more detailed with HILIC-simplified fractions to the point where hundreds of signals are present and 2D NMR correlations permit significant structural identifications.

Interactions of poly(amidoamine) dendrimers with human serum albumin: binding constants and mechanisms.

The interactions of nanomaterials with plasma proteins have a significant impact on their in vivo transport and fate in biological fluids. This article discusses the binding of human serum albumin (HSA) to poly(amidoamine) [PAMAM] dendrimers. We use protein-coated silica particles to measure the HSA binding constants (K(b)) of a homologous series of 19 PAMAM dendrimers in aqueous solutions at physiological pH (7.4) as a function of dendrimer generation, terminal group, and core chemistry. To gain insight into the mechanisms of HSA binding to PAMAM dendrimers, we combined (1)H NMR, saturation transfer difference (STD) NMR, and NMR diffusion ordered spectroscopy (DOSY) of dendrimer-HSA complexes with atomistic molecular dynamics (MD) simulations of dendrimer conformation in aqueous solutions. The binding measurements show that the HSA binding constants (K(b)) of PAMAM dendrimers depend on dendrimer size and terminal group chemistry. The NMR (1)H and DOSY experiments indicate that the interactions between HSA and PAMAM dendrimers are relatively weak. The (1)H NMR STD experiments and MD simulations suggest that the inner shell protons of the dendrimers groups interact more strongly with HSA proteins. These interactions, which are consistently observed for different dendrimer generations (G0-NH(2)vs G4-NH(2)) and terminal groups (G4-NH(2)vs G4-OH with amidoethanol groups), suggest that PAMAM dendrimers adopt backfolded configurations as they form weak complexes with HSA proteins in aqueous solutions at physiological pH (7.4).

Evaluation of sample preparation methods for nuclear magnetic resonance metabolic profiling studies with Eisenia fetida

The earthworm Eisenia fetida is frequently used in ecotoxicological studies; however, it has not yet been investigated using proton nuclear magnetic resonance ((1)H NMR) metabolic profiling methods. The present study investigates the impact of depuration time, sample homogenization, and different extraction solvents on the quality and reproducibility of the (1)H NMR spectra of E. fetida with the goal of determining whether this species is suitable for future metabonomic studies. A depuration time of 96 h, followed by intact lyophilization before homogenization and extraction into a deuterium oxide (D(2)O)-based phosphate buffer, was found to produce extracts with excellent (1)H NMR reproducibility. The D(2)O buffer extracted the largest quantity of the widest variety of earthworm metabolites, which is consistent with the results from other studies using different earthworm species. Nuclear magnetic resonance assignments of the major metabolites in the D(2)O-based buffer also were performed and found to be similar to those for other earthworm species, such as Eisenia veneta, but also to have characteristic attributes in E. fetida. The major metabolites identified include amino acids (alanine, arginine, glutamic acid, glutamine, glycine, leucine, lysine, phenylalanine, serine, tyrosine, and valine), two sugars (glucose and maltose), the sugar alcohol mannitol, and the polyalcohol inositol. Two other earthworm species (Lumbricus rubellus and Lumbricus terrestris) also were examined using protocols developed for E. fetida, and of the three species, the (1)H NMR spectra of E. fetida had the least variation, indicating this species is well-suited for future metabolomic-based ecotoxicity studies.

The Chemical Ecology of Soil Organic Matter Molecular Constituents

Soil organic matter (OM) contains vast stores of carbon, and directly supports microbial, plant, and animal life by retaining essential nutrients and water in the soil. Soil OM plays important roles in biological, chemical, and physical processes within the soil, and arguably plays a major role in maintaining long-term ecological stability in a changing world. Despite its importance, there is a great deal still unknown about soil OM chemical ecology. The development of sophisticated analytical methods have reshaped our understanding of soil OM composition, which is now believed to be comprised of plant and microbial products at various stages of decomposition. The methods also have recently been applied to study environmental change in various settings and have provided unique insight with respect to soil OM chemical ecology. The goal of this review is to highlight the methods used to characterize soil OM structure, source, and degradation that have enabled precise observations of OM and associated ecological shifts. Although the chemistry of soil OM is important in its overall fate in ecosystems, the studies conducted to date suggest that ecological function is not defined by soil OM chemistry alone. The long-standing questions regarding soil OM stability and recalcitrance will likely be answered when several molecular methods are used in tandem to closely examine structure, source, age, degradation stage, and interactions of specific OM components in soil.

1H NMR metabolomics of earthworm exposure to sub-lethal concentrations of phenanthrene in soil

1H NMR metabolomics was used to monitor earthworm responses to sub-lethal (50-1500 mg/kg) phenanthrene exposure in soil. Total phenanthrene was analyzed via soxhlet extraction, bioavailable phenanthrene was estimated by hydroxypropyl-beta-cyclodextrin (HPCD) and 1-butanol extractions and sorption to soil was assessed by batch equilibration. Bioavailable phenanthrene (HPCD-extracted) comprised approximately 65-97% of total phenanthrene added to the soil. Principal component analysis (PCA) showed differences in responses between exposed earthworms and controls after 48 h exposure. The metabolites that varied with exposure included amino acids (isoleucine, alanine and glutamine) and maltose. PLS models indicated that earthworm response is positively correlated to both total phenanthrene concentration and bioavailable (HPCD-extracted) phenanthrene in a freshly spiked, unaged soil. These results show that metabolomics is a powerful, direct technique that may be used to monitor contaminant bioavailability and toxicity of sub-lethal concentrations of contaminants in the environment. These initial findings warrant further metabolomic studies with aged contaminated soils.

Earthworm Sublethal Responses to Titanium Dioxide Nanomaterial in Soil Detected by 1H NMR Metabolomics

¹H NMR-based metabolomics was used to examine the response of Eisenia fetida earthworms raised from juveniles for 20-23 weeks in soil spiked with either 20 or 200 mg/kg of a commercially available uncoated titanium dioxide (TiO(2)) nanomaterial (nominal diameter of 5 nm). To distinguish responses specific to particle size, soil treatments spiked with a micrometer-sized TiO(2) material (nominal diameter, <45 μm) at the same concentrations (20 and 200 mg/kg) were also included in addition to an unspiked control soil. Multivariate statistical analysis of the (1)H NMR spectra for aqueous extracts of E. fetida tissue suggested that earthworms exhibited significant changes in their metabolic profile following TiO(2) exposure for both particle sizes. The observed earthworm metabolic changes appeared to be consistent with oxidative stress, a proposed mechanism of toxicity for nanosized TiO(2). In contrast, a prior study had observed no impairment of E. fetida survival, reproduction, or growth following exposure to the same TiO(2) spiked soils. This suggests that (1)H NMR-based metabolomics provides a more sensitive measure of earthworm response to TiO(2) materials in soil and that further targeted assays to detect specific cellular or molecular level damage to earthworms caused by chronic exposure to TiO(2) are warranted.

Arctic Permafrost Active Layer Detachments Stimulate Microbial Activity and Degradation of Soil Organic Matter

Large quantities of soil organic carbon in Arctic permafrost zones are becoming increasingly unstable due to a warming climate. High temperatures and substantial rainfall in July 2007 in the Canadian High Arctic resulted in permafrost active layer detachments (ALDs) that redistributed soils throughout a small watershed in Nunavut, Canada. Molecular biomarkers and NMR spectroscopy were used to measure how ALDs may lead to microbial activity and decomposition of previously unavailable soil organic matter (SOM). Increased concentrations of extracted bacterial phospholipid fatty acids (PLFAs) and large contributions from bacterial protein/peptides in the NMR spectra at recent ALDs suggest increased microbial activity. PLFAs were appreciably depleted in a soil sample where ALDs occurred prior to 2003. However an enrichment of bacterial derived peptidoglycan was observed by (1)H-(13)C heteronuclear multiple quantum coherence (HMQC) and (1)H diffusion edited (DE) NMR and enhanced SOM degradation was observed by (13)C solid-state NMR. These data suggest that a previous rise in microbial activity, as is currently underway at the recent ALD site, led to degradation and depletion of labile SOM components. Therefore, this study indicates that ALDs may amplify climate change due to the release of labile SOM substrates from thawing High Arctic permafrost.

Comprehensive multiphase NMR spectroscopy: Basic experimental approaches to differentiate phases in heterogeneous samples

Heterogeneous samples, such as soils, sediments, plants, tissues, foods and organisms, often contain liquid-, gel- and solid-like phases and it is the synergism between these phases that determine their environmental and biological properties. Studying each phase separately can perturb the sample, removing important structural information such as chemical interactions at the gel-solid interface, kinetics across boundaries and conformation in the natural state. In order to overcome these limitations a Comprehensive Multiphase-Nuclear Magnetic Resonance (CMP-NMR) probe has been developed, and is introduced here, that permits all bonds in all phases to be studied and differentiated in whole unaltered natural samples. The CMP-NMR probe is built with high power circuitry, Magic Angle Spinning (MAS), is fitted with a lock channel, pulse field gradients, and is fully susceptibility matched. Consequently, this novel NMR probe has to cover all HR-MAS aspects without compromising power handling to permit the full range of solution-, gel- and solid-state experiments available today. Using this technology, both structures and interactions can be studied independently in each phase as well as transfer/interactions between phases within a heterogeneous sample. This paper outlines some basic experimental approaches using a model heterogeneous multiphase sample containing liquid-, gel- and solid-like components in water, yielding separate (1)H and (13)C spectra for the different phases. In addition, (19)F performance is also addressed. To illustrate the capability of (19)F NMR soil samples, containing two different contaminants, are used, demonstrating a preliminary, but real-world application of this technology. This novel NMR approach possesses a great potential for the in situ study of natural samples in their native state.

Determining the molecular interactions of perfluorinated carboxylic acids with human sera and isolated human serum albumin using nuclear magnetic resonance spectroscopy

Perfluorooctanoate (PFOA) is ubiquitous in North American human sera and has a serum half-life of 3.5 years in humans. The molecular interactions that lead to the bioaccumulation of these hydrophobic and lipophobic molecules in human blood are not well understood. Perfluorohexanoic acid (PFHxA) and PFOA were used as model perfluorinated carboxylic acids (PFCAs) to characterize the major site of PFCA interaction in human sera. Using novel heteronuclear saturation transfer difference nuclear magnetic resonance spectroscopy experiments, human serum albumin (HSA) was identified as the major site of interaction for both PFHxA and PFOA in human sera. Heteronuclear single quantum coherence nuclear magnetic resonance experiments were then performed to interrogate site-specific interactions of PFHxA and PFOA with isolated HSA. Perfluorohexanoic acid was found to bind specifically to Sudlows drug-binding site II, whereas PFOA interacted preferentially with Sudlows drug-binding site I at the lower concentration, with additional interactions developing at the higher concentration. These experiments highlight the utility of nuclear magnetic resonance spectrometry as a tool to observe the in situ interactions of chemical contaminants with biological systems. Both PFCAs displaced the endogenous HSA ligand oleic acid at concentrations lower than observed for the drugs ibuprofen and phenylbutazone, which are established HSA ligands. Interactions between PFCAs and HSA may affect the pharmacokinetics and distribution of fatty acids and certain drugs in the human body and warrant further investigation.

1H NMR-based metabolomics investigation of Daphnia magna responses to sub-lethal exposure to arsenic, copper and lithium

Metal and metalloid contamination constitutes a major concern in aquatic ecosystems. Thus it is important to find rapid and reliable indicators of metal stress to aquatic organisms. In this study, we tested the use of (1)H nuclear magnetic resonance (NMR) - based metabolomics to examine the response of Daphnia magna neonates after a 48h exposure to sub-lethal concentrations of arsenic (49μgL(-1)), copper (12.4μgL(-1)) or lithium (1150μgL(-1)). Metabolomic responses for all conditions were compared to a control using principal component analysis (PCA) and metabolites that contributed to the variation between the exposures and the control condition were identified and quantified. The PCA showed that copper and lithium exposures result in statistically significant metabolite variations from the control. Contributing to this variation was a number of amino acids such as: phenylalanine, leucine, lysine, glutamine, glycine, alanine, methionine and glutamine as well as the nucleobase uracil and osmolyte glycerophosphocholine. The similarities in metabolome changes suggest that lithium has an analogous mode of toxicity to that of copper, and may be impairing energy production and ionoregulation. The PCA also showed that arsenic exposure resulted in a metabolic shift in comparison to the control population but this change was not statistically significant. However, significant changes in specific metabolites such as alanine and lysine were observed, suggesting that energy metabolism is indeed disrupted. This research demonstrates that (1)H NMR-based metabolomics is a viable platform for discerning metabolomic changes and mode of toxicity of D. magna in response to metal stressors in the environment.