Hyperpolarization-Enhanced NMR Spectroscopy of Unaltered Biofluids Using Photo-CIDNP.

The direct and unambiguous detection and identification of individual metabolite molecules present in complex biological mixtures constitute a major challenge in (bio)analytical research. In this context, nuclear magnetic resonance (NMR) spectroscopy has proven to be particularly powerful owing to its ability to provide both qualitative and quantitative atomic-level information on multiple analytes simultaneously in a noninvasive manner. Nevertheless, NMR suffers from a low inherent sensitivity and, moreover, lacks selectivity regarding the number of individual analytes to be studied in a mixture of a myriad of structurally and chemically very different molecules, e.g., metabolites in a biofluid. Here, we describe a method that circumvents these shortcomings via performing selective, photochemically induced dynamic nuclear polarization (photo-CIDNP) enhanced NMR spectroscopy on unmodified complex biological mixtures, i.e., human urine and serum, which yields a single, background-free one-dimensional NMR spectrum. In doing this, we demonstrate that photo-CIDNP experiments on unmodified complex mixtures of biological origin are feasible, can be performed straightforwardly in the native aqueous medium at physiological metabolite concentrations, and act as a spectral filter, facilitating the analysis of NMR spectra of complex biofluids. Due to its noninvasive nature, the method is fully compatible with state-of-the-art metabolomic protocols providing direct spectroscopic information on a small, carefully selected subset of clinically relevant metabolites. We anticipate that this approach, which, in addition, can be combined with existing high-throughput/high-sensitivity NMR methodology, holds great promise for further in-depth studies and development for use in metabolomics and many other areas of analytical research.

Different profiles of soil phosphorous compounds depending on tree species and availability of soil phosphorus in a tropical rainforest in French Guiana.

BACKGROUND: The availability of soil phosphorus (P) often limits the productivities of wet tropical lowland forests. Little is known, however, about the metabolomic profile of different chemical P compounds with potentially different uses and about the cycling of P and their variability across space under different tree species in highly diverse tropical rainforests. RESULTS: We hypothesised that the different strategies of the competing tree species to retranslocate, mineralise, mobilise, and take up P from the soil would promote distinct soil 31P profiles. We tested this hypothesis by performing a metabolomic analysis of the soils in two rainforests in French Guiana using 31P nuclear magnetic resonance (NMR). We analysed 31P NMR chemical shifts in soil solutions of model P compounds, including inorganic phosphates, orthophosphate mono- and diesters, phosphonates, and organic polyphosphates. The identity of the tree species (growing above the soil samples) explained > 53% of the total variance of the 31P NMR metabolomic profiles of the soils, suggesting species-specific ecological niches and/or species-specific interactions with the soil microbiome and soil trophic web structure and functionality determining the use and production of P compounds. Differences at regional and topographic levels also explained some part of the the total variance of the 31P NMR profiles, although less than the influence of the tree species. Multivariate analyses of soil 31P NMR metabolomics data indicated higher soil concentrations of P biomolecules involved in the active use of P (nucleic acids and molecules involved with energy and anabolism) in soils with lower concentrations of total soil P and higher concentrations of P-storing biomolecules in soils with higher concentrations of total P. CONCLUSIONS: The results strongly suggest "niches" of soil P profiles associated with physical gradients, mostly topographic position, and with the specific distribution of species along this gradient, which is associated with species-specific strategies of soil P mineralisation, mobilisation, use, and uptake.

Engineering the synthetic ß-alanine pathway in Komagataella phaffii for conversion of methanol into 3-hydroxypropionic acid.

BACKGROUND: Methanol is increasingly gaining attraction as renewable carbon source to produce specialty and commodity chemicals, as it can be generated from renewable sources such as carbon dioxide (CO2). In this context, native methylotrophs such as the yeast Komagataella phaffii (syn Pichia pastoris) are potentially attractive cell factories to produce a wide range of products from this highly reduced substrate. However, studies addressing the potential of this yeast to produce bulk chemicals from methanol are still scarce. 3-Hydroxypropionic acid (3-HP) is a platform chemical which can be converted into acrylic acid and other commodity chemicals and biopolymers. 3-HP can be naturally produced by several bacteria through different metabolic pathways. RESULTS: In this study, production of 3-HP via the synthetic ß-alanine pathway has been established in K. phaffii for the first time by expressing three heterologous genes, namely panD from Tribolium castaneum, yhxA from Bacillus cereus, and ydfG from Escherichia coli K-12. The expression of these key enzymes allowed a production of 1.0 g l-1 of 3-HP in small-scale cultivations using methanol as substrate. The addition of a second copy of the panD gene and selection of a weak promoter to drive expression of the ydfG gene in the PpCß21 strain resulted in an additional increase in the final 3-HP titer (1.2 g l-1). The 3-HP-producing strains were further tested in fed-batch cultures. The best strain (PpCß21) achieved a final 3-HP concentration of 21.4 g l-1 after 39 h of methanol feeding, a product yield of 0.15 g g-1, and a volumetric productivity of 0.48 g l-1 h-1. Further engineering of this strain aiming at increasing NADPH availability led to a 16% increase in the methanol consumption rate and 10% higher specific productivity compared to the reference strain PpCß21. CONCLUSIONS: Our results show the potential of K. phaffii as platform cell factory to produce organic acids such as 3-HP from renewable one-carbon feedstocks, achieving the highest volumetric productivities reported so far for a 3-HP production process through the ß-alanine pathway.

Fatty Acids and Metabolomic Composition of Follicular Fluid Collected from Environments Associated with Good and Poor Oocyte Competence in Goats.

In goats, embryo oocyte competence is affected by follicle size regardless the age of the females. In previous studies we have found differences in blastocyst development between oocytes coming of small (<3 mm) and large follicles (>3 mm) in prepubertal (1−2 months-old) goats. Oocyte competence and Follicular Fluid (FF) composition changes throughout follicle growth. The aim of this study was to analyze Fatty Acids (FAs) composition and metabolomic profiles of FF recovered from small and large follicles of prepubertal goats and follicles of adult goats. FAs were analyzed by chromatography and metabolites by 1H-Nuclear Magnetic Resonance (1H-NMR) Spectrometry. The results showed important differences between adult and prepubertal follicles: (a) the presence of α,ß-glucose in adult and no detection in prepubertal; (b) lactate, -N-(CH3)3 groups and inositol were higher in prepubertal (c) the percentage of Linolenic Acid, Total Saturated Fatty Acids and n-3 PUFAs were higher in adults; and (d) the percentage of Linoleic Acid, total MUFAs, PUFAs, n-6 PUFAs and n-6 PUFAs: n-3 PUFAs ratio were higher in prepubertal goats. Not significant differences were found in follicle size of prepubertal goats, despite the differences in oocyte competence for in vitro embryo production.

Special Issue: NMR-Based Metabolomics.

Nuclear magnetic resonance (NMR) spectroscopy remains one of the core analytical platforms for metabolomics, providing complementary chemical information to others, such as mass spectrometry, and offering particular advantages in some areas of research on account of its inherent robustness, reproducibility, and phenomenal dynamic range [...].

31P-NMR Metabolomics Revealed Species-Specific Use of Phosphorous in Trees of a French Guiana Rainforest.

Productivity of tropical lowland moist forests is often limited by availability and functional allocation of phosphorus (P) that drives competition among tree species and becomes a key factor in determining forestall community diversity. We used non-target 31P-NMR metabolic profiling to study the foliar P-metabolism of trees of a French Guiana rainforest. The objective was to test the hypotheses that P-use is species-specific, and that species diversity relates to species P-use and concentrations of P-containing compounds, including inorganic phosphates, orthophosphate monoesters and diesters, phosphonates and organic polyphosphates. We found that tree species explained the 59% of variance in 31P-NMR metabolite profiling of leaves. A principal component analysis showed that tree species were separated along PC 1 and PC 2 of detected P-containing compounds, which represented a continuum going from high concentrations of metabolites related to non-active P and P-storage, low total P concentrations and high N:P ratios, to high concentrations of P-containing metabolites related to energy and anabolic metabolism, high total P concentrations and low N:P ratios. These results highlight the species-specific use of P and the existence of species-specific P-use niches that are driven by the distinct species-specific position in a continuum in the P-allocation from P-storage compounds to P-containing molecules related to energy and anabolic metabolism.

Simultaneous Enantiospecific Detection of Multiple Compounds in Mixtures using NMR Spectroscopy.

Chirality plays a fundamental role in nature, but its detection and quantification still face many limitations. To date, the enantiospecific analysis of mixtures necessarily requires prior separation of the individual components. The simultaneous enantiospecific detection of multiple chiral molecules in a mixture represents a major challenge, which would lead to a significantly better understanding of the underlying biological processes; for example, via enantiospecifically analysing metabolites in their native environment. Here, we report on the first in situ enantiospecific detection of a thirty-nine-component mixture. As a proof of concept, eighteen essential amino acids at physiological concentrations were simultaneously enantiospecifically detected using NMR spectroscopy and a chiral solvating agent. This work represents a first step towards the simultaneous multicomponent enantiospecific analysis of complex mixtures, a capability that will have substantial impact on metabolism studies, metabolic phenotyping, chemical reaction monitoring, and many other fields where complex mixtures containing chiral molecules require efficient characterisation.

Interleaved Dual NMR Acquisition of Equivalent Transfer Pathways in TOCSY and HSQC Experiments.

A dual NMR data acquisition strategy to handle and detect two active equivalent transfer pathways is presented and discussed. We illustrate the power of this time-efficient approach by collecting two different 2D spectra simultaneously in a single experiment: i) TOCSY or HSQC-TOCSY spectra with different mixing times, ii) F2-13 C-coupled and decoupled HSQC spectra, iii) conventional and pure-shift HSQC spectra, or iv) complementary HSQC and HSQC-TOCSY spectra.

Chiral Recognition by Dissolution DNP NMR Spectroscopy of 13C-Labeled dl-Methionine.

A method based on d-DNP NMR spectroscopy to study chiral recognition is described for the first time. The enantiodifferentiation of a racemic metabolite in a millimolar aqueous solution using a chiral solvating agent was performed. Hyperpolarized 13C-labeled dl-methionine enantiomers were differently observed with a single-scan 13C NMR experiment, while the chiral auxiliary at thermal equilibrium remained unobserved. The method developed entails a step forward in the chiral recognition of small molecules by NMR spectroscopy, opening new possibilities in situations where the sensitivity is limited, for example, when a low concentration of analyte is available or when the measurement of an insensitive nucleus, like 13C, is required. The advantages and current limitations of the method, as well as future perspectives, are discussed.

Direct Monitoring of Exogenous γ-Hydroxybutyric Acid in Body Fluids by NMR Spectroscopy.

γ-Hydroxybutyric acid (GHB) is a popular drug increasingly associated with cases of drug-facilitated sexual assault (DFSA). Currently, expanding procedures of analysis and having forensic evidence of GHB intake in a long term are mandatory. Up to now, most studies have been performed using GC/MS and LC-MS as analytical platforms, which involve significant manipulation of the sample and, often, indirect measurements. In this work, procedures used in NMR-based metabolomics were applied to a GHB clinical trial on urine and serum. Detection, identification, and quantification of the drug by NMR methods were surveyed, as well as the use of NMR-based metabolomics for the search of potential surrogate biomarkers of GHB consumption. Results demonstrated the suitability of NMR spectroscopy, as a robust nondestructive technique, to fast and directly monitor (detect, identify, and quantify) exogenous GHB in almost intact body fluids and its high potential in the search for metabolites associated with GHB intake.

Shifts in plant foliar and floral metabolomes in response to the suppression of the associated microbiota.

BACKGROUND: The phyllospheric microbiota is assumed to play a key role in the metabolism of host plants. Its role in determining the epiphytic and internal plant metabolome, however, remains to be investigated. We analyzed the Liquid Chromatography-Mass Spectrometry (LC-MS) profiles of the epiphytic and internal metabolomes of the leaves and flowers of Sambucus nigra with and without external antibiotic treatment application. RESULTS: The epiphytic metabolism showed a degree of complexity similar to that of the plant organs. The suppression of microbial communities by topical applications of antibiotics had a greater impact on the epiphytic metabolome than on the internal metabolomes of the plant organs, although even the latter changed significantly both in leaves and flowers. The application of antibiotics decreased the concentration of lactate in both epiphytic and organ metabolomes, and the concentrations of citraconic acid, acetyl-CoA, isoleucine, and several secondary compounds such as terpenes and phenols in the epiphytic extracts. The metabolite pyrogallol appeared in the floral epiphytic community only after the treatment. The concentrations of the amino acid precursors of the ketoglutarate-synthesis pathway tended to decrease in the leaves and to increase in the foliar epiphytic extracts. CONCLUSIONS: These results suggest that anaerobic and/or facultative anaerobic bacteria were present in high numbers in the phyllosphere and in the apoplasts of S. nigra. The results also show that microbial communities play a significant role in the metabolomes of plant organs and could have more complex and frequent mutualistic, saprophytic, and/or parasitic relationships with internal plant metabolism than currently assumed.

Warming differentially influences the effects of drought on stoichiometry and metabolomics in shoots and roots.

Plants in natural environments are increasingly being subjected to a combination of abiotic stresses, such as drought and warming, in many regions. The effects of each stress and the combination of stresses on the functioning of shoots and roots have been studied extensively, but little is known about the simultaneous metabolome responses of the different organs of the plant to different stresses acting at once. We studied the shift in metabolism and elemental composition of shoots and roots of two perennial grasses, Holcus lanatus and Alopecurus pratensis, in response to simultaneous drought and warming. These species responded differently to individual and simultaneous stresses. These responses were even opposite in roots and shoots. In plants exposed to simultaneous drought and warming, terpenes, catechin and indole acetic acid accumulated in shoots, whereas amino acids, quinic acid, nitrogenous bases, the osmoprotectants choline and glycine betaine, and elements involved in growth (nitrogen, phosphorus and potassium) accumulated in roots. Under drought, warming further increased the allocation of primary metabolic activity to roots and changed the composition of secondary metabolites in shoots. These results highlight the plasticity of plant metabolomes and stoichiometry, and the different complementary responses of shoots and roots to complex environmental conditions.

Strong relationship between elemental stoichiometry and metabolome in plants.

Shifts in the elemental stoichiometry of organisms in response to their ontogeny and to changing environmental conditions should be related to metabolomic changes because elements operate mostly as parts of molecular compounds. Here we show this relationship in leaves of Erica multiflora throughout their seasonal development and in response to moderate experimental field conditions of drought and warming. The N/P ratio in leaves decreased in the metabolically active growing seasons, coinciding with an increase in the content of primary metabolites. These results support the growth-rate hypothesis that states that rapidly growing organisms present low N/P ratios because of the increase in allocation of P to RNA. The foliar N/K and P/K ratios were lower in summer and in the drought treatment, in accordance with the role of K in osmotic protection, and coincided with the increase of compounds related to the avoidance of water stress. These results provide strong evidence of the relationship between the changes in foliar C/N/P/K stoichiometry and the changes in the leaf's metabolome during plant growth and environmental stress. Thus these results represent a step in understanding the relationships between stoichiometry and an organism's lifestyle.

Drought enhances folivory by shifting foliar metabolomes in Quercus ilex trees.

At the molecular level, folivory activity on plants has mainly been related to the foliar concentrations of nitrogen (N) and/or particular metabolites. We studied the responses of different nutrients and the whole metabolome of Quercus ilex to seasonal changes and to moderate field experimental conditions of drought, and how this drought may affect folivory activity, using stoichiometric and metabolomic techniques. Foliar potassium (K) concentrations increased in summer and consequently led to higher foliar K : phosphorus (P) and lower carbon (C) : K and N : K ratios. Foliar N : P ratios were not lowest in spring as expected by the growth rate hypothesis. Trees exposed to moderate drought presented higher concentrations of total sugars and phenolics and these trees also experienced more severe folivory attack. The foliar increases in K, sugars and antioxidant concentrations in summer, the driest Mediterranean season, indicated enhanced osmoprotection under natural drought conditions. Trees under moderate drought also presented higher concentrations of sugars and phenolics; a plant response to avoid water loss. These shifts in metabolism produced an indirect relationship between increased drought and folivory activity.

Enantiodifferentiation through frequency-selective pure-shift (1)H nuclear magnetic resonance spectroscopy.

A frequency-selective 1D (1) H nuclear magnetic resonance (NMR) experiment for the fast and sensitive determination of chemical-shift differences between overlapped resonances is proposed. The resulting fully homodecoupled (1) H NMR resonances appear as resolved 1D singlets without their typical J(HH) coupling constant multiplet structures. The high signal dispersion that is achieved is then exploited in enantiodiscrimination studies by using chiral solvating agents.

Targeted and untargeted serum NMR metabolomics to reveal initial kidney disease in diabetes mellitus.

Serum 1H NMR metabolomics has been used as a diagnostic tool for screening type 2 diabetes (T2D) with chronic kidney disease (CKD) as comorbidity. This work aimed to evaluate 1H NMR data to detect the initial kidney damage and CKD in T2D subjects, through multivariate statistical analysis. Clinical data and biochemical parameters were obtained for classifying five experimental groups using KDIGO guidelines: Control (healthy subjects), T2D, T2D-CKD-mild, T2D-CKD-moderate, and T2D-CKD-severe. Serum 1H NMR spectra were recorded to follow two strategies: one based on metabolite-to-creatinine (Met/Cr) ratios as targeted metabolomics, and the second one based on untargeted metabolomics from the 1H NMR profile. A prospective biomarkers panel of the early stage of T2D-CKD based in metabolite-to-creatinine ratio (ornithine/Cr, serine/Cr, mannose/Cr, acetate/Cr, acetoacetate/Cr, formate/Cr, and glutamate/Cr) was proposed. Later, a statistical model based on non-targeted metabolomics was used to predict initial CKD, and its metabolic pathway analysis allowed identifying the most affected pathways: phenylalanine, tyrosine, and tryptophan biosynthesis; valine, leucine, and isoleucine degradation; glyoxylate and dicarboxylate metabolism; glycine, serine, and threonine metabolism; and histidine metabolism. Nonetheless, further studies with a larger cohort are advised to precise ranges in metabolite-to-creatinine ratios and evaluate the prediction pertinency to detect initial CKD in T2D patients in both statistical models proposed.

13C NMR spectroscopy for the differentiation of enantiomers using chiral solvating agents.

The utility of (13)C NMR spectroscopy for the differentiation of enantiomers using chiral solvating agents (CSA) is stated. Three examples involving the enantiodifferentiation of a drug, a metabolite and a reactant in aqueous and organic solutions have been chosen to show it. The intrinsic high dispersion of (13)C nucleus, as well as the singlet nature of the signals in standard experiments makes (13)C NMR experiments a powerful alternative or complement to (1)H NMR experiments; specially, when studying pure compounds with complex proton spectra or mixtures of compounds, as in chiral metabonomics, where severe overlapping exists in proton spectrum. To evaluate and compare the quality of the enantioresolution of a NMR signal we introduce the enantiodifferentiation quotient, E, that considers the complexity of (1)H multiplets (and in general the width) of the original signal. It has been observed that the error in the measurement of the enantiomeric molar ratio can be related to the E value. The sensitivity and experimental time of a wide range of chiral analyte concentrations were also assessed.

Chiral metabonomics: 1H NMR-based enantiospecific differentiation of metabolites in human urine via direct cosolvation with ß-cyclodextrin.

Differences in molecular chirality remain an important issue in drug metabolism and pharmacokinetics for the pharmaceutical industry and regulatory authorities, and chirality is an important feature of many endogenous metabolites. We present a method for the rapid, direct differentiation and identification of chiral drug enantiomers in human urine without pretreatment of any kind. Using the well-known anti-inflammatory chemical ibuprofen as one example, we demonstrate that the enantiomers of ibuprofen and the diastereoisomers of one of its main metabolites, the glucuronidated carboxylate derivative, can be resolved by (1)H NMR spectroscopy as a consequence of direct addition of the chiral cosolvating agent (CSA) ß-cyclodextrin (ßCD). This approach is simple, rapid, and robust, involves minimal sample manipulation, and does not require derivatization or purification of the sample. In addition, the method should allow the enantiodifferentiation of endogenous chiral metabolites, and this has potential value for differentiating metabolites from mammalian and microbial sources in biofluids. From these initial findings, we propose that more extensive and detailed enantiospecific metabolic profiling could be possible using CSA-NMR spectroscopy than has been previously reported.

Cyclopropanation of α-mycolic acids is not required for cording in Mycobacterium brumae and Mycobacterium fallax.

The capacity to form microscopic cords (cording) of Mycobacterium species has been related to their virulence. The compounds responsible for cording are unknown, but a recent study has shown that cording could be related to the fine structure of α-mycolic acids. This investigation attributes the need for a proximal cyclopropane in α-mycolic acids for cording in Mycobacterium tuberculosis and Mycobacterium bovis BCG and proposes cyclopropanases as good targets for new chemotherapeutic agents. As other Mycobacterium species in addition to M. tuberculosis and M. bovis form microscopic cords, it would be of major interest to know whether the relationship between proximal cyclopropanation of α-mycolic acids and cording could be extended to non-tuberculous mycobacteria. In this study, we have examined the correlation between the cording and cyclopropanation of α-mycolic acids in two species, Mycobacterium brumae and Mycobacterium fallax. Scanning electron microscopy images showed, for the first time to our knowledge, the fine structure of microscopic cords of M. brumae and M. fallax, confirming that these two species form true cords. Furthermore, NMR analysis performed on the same cording cultures corroborates the absence of cyclopropane rings in their α-mycolic acids. Therefore, we can conclude that the correlation between cording and cyclopropanation of α-mycolic acids cannot be extended to all mycobacteria. As M. brumae and M. fallax grow rapidly and have a simple pattern of mycolic acids (only α-unsaturated mycolic acids), we propose these two species as suitable models for the study of the role of mycolic acids in cording.