Nuclear Magnetic Resonance Metabolic Profiling Research Articles

NMR metabolic profile of human follicular fluid

The environment of the oocyte during its in vivo maturation consists of follicular fluid (FF) and is surrounded by granulosa cells. The FF is derived from the sanguineous plasma and secretions, synthesised in the follicle wall, that contain a large variety of growth factors, cytokines, amino acids, and other metabolites. These metabolites are presumably involved in the physiology of the oocyte. The identification, quantification and study of FF metabolites can provide additional information about the oocyte state which can be helpful in distinguishing those oocytes that have a greater capacity to be fertilised and to develop properly. The aim of this work is to identify the metabolic profile of FF samples exhaustively using High Resolution Nuclear Magnetic Resonance (NMR). A total of 30 FF samples from oocyte donors (<35 years) were analysed. Different monodimensional (1D) and bidimensional (2D) (homo and heteronuclear) NMR experiments were acquired. A total of 131 chemical shifts were assigned and 42 metabolites, including as example glucose, lactate, acetate, acetoacetate, pyruvate and beta-hydroxybutyrate, were identified. High correlations were found between these important intermediaries of the energetic metabolic pathways of the follicle which can indicate the importance of these pathways in oocyte development. Some of these identified metabolites might be useful as biomarkers of the follicular maturation state, allowing oocytes with a higher fertilisation potential to be selected, thereby increasing pregnancy rates in women following in vitro fertilisation (IVF) treatments.

The Influence of a Sports Drink on the Postexercise Metabolism of Elite Athletes as Investigated by NMR-Based Metabolomics

Objective: The aim of this study is to evaluate the systemic effects of an isotonic sports drink on the metabolic status of athletes of the Italian Olympic rowing team during recovery after strenuous and prolonged physical exercise by means of nuclear magnetic resonance (NMR)-based metabolomics analysis on plasma and urine.Methods: Forty-four male athletes of the Italian Olympic rowing team were enrolled in a double-blind crossover study. All subjects underwent 2 evaluations at 1-week intervals. The evaluation was performed on a rowing ergometer after strenuous physical exercise to produce a state of dehydration. Afterward, the athletes were rehydrated either with a green tea–based carbohydrate-hydroelectrolyte drink or with oligomineral water. Three blood samples were drawn for each subject: at rest, after the exercise, and following rehydratation, while 2 urine samples were collected: at rest and after the rehydratation period. Biofluid samples were analyzed by high-resolution 1H NMR metabolic profiling combined with multilevel simultaneous data-analysis (MSCA) and partial-least squares-discriminant analysis (PLS-DA).Results: The between-subject variations, as evaluated by MSCA, reflected the variations of lactate levels induced by the physical exercise. Analysis of the within-individual variance using multilevel PLS-DA models of plasma and urine metabolic profiles showed an effect of the green tea–based sports drink on glucose, citrate, and lactate levels in plasma and on acetone, 3-OH-butyrate, and lactate levels in urine. The increase of caffeine and hippuric acid levels in urine indicated the absorption of green tea extract components.Conclusions: NMR-based metabolomics allowed the complex effects of a green tea extract–based carbohydrate/hydroelectrolyte beverage on the energy metabolism of athletes during recovery by postexercise rehydration to be evaluated.

NMR metabolic profiling of organic and aqueous sea bass extracts: Implications in the discrimination of wild and cultured sea bass

The nuclear magnetic resonance (NMR) technique was used as analytical tool to determine the complete metabolic profiling of sea bass extracts: water-soluble metabolites belonging to different classes such as sugars, amino acids, dipeptides and organic acids as well as metabolites soluble in organic solvent such as lipids, sterols and fatty acids were identified. The metabolite profiling together with a suitable statistical analysis were used to discriminate between wild and cultured sea bass samples. Preliminary results show that discrimination between wild and cultured sea bass was obtained not only using fatty acid composition but also cholesterol and phosphatidylethanolamine and some water-soluble metabolites such as choline, trimethylamine oxide, glutamine, fumaric and malic acids.

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.

Transcriptional and metabolic adjustments in ADP-glucose pyrophosphorylase-deficient bt2 maize kernels.

During the cloning of monogenic recessive mutations responsible for a defective kernel phenotype in a Mutator-induced Zea mays mutant collection, we isolated a new mutant allele in Brittle2 (Bt2), which codes for the small subunit of ADP-glucose pyrophosphorylase (AGPase), a key enzyme in starch synthesis. Reverse transcription-polymerase chain reaction experiments with gene-specific primers confirmed a predominant expression of Bt2 in endosperm, of Agpsemzm in embryo, and of Agpslzm in leaf, but also revealed considerable additional expression in various tissues for all three genes. Bt2a, the classical transcript coding for a cytoplasmic isoform, was almost exclusively expressed in the developing endosperm, whereas Bt2b, an alternative transcript coding for a plastidial isoform, was expressed in almost all tissues tested with a pattern very similar to that of Agpslzm. The phenotypic analysis showed that, at 30 d after pollination (DAP), mutant kernels were plumper than wild-type kernels, that the onset of kernel collapse took place between 31 and 35 DAP, and that the number of starch grains was greatly reduced in the mutant endosperm but not the mutant embryo. A comparative transcriptome analysis of wild-type and bt2-H2328 kernels at middevelopment (35 DAP) with the 18K GeneChip Maize Genome Array led to the conclusion that the lack of Bt2-encoded AGPase triggers large-scale changes on the transcriptional level that concern mainly genes involved in carbohydrate or amino acid metabolic pathways. Principal component analysis of (1)H nuclear magnetic resonance metabolic profiles confirmed the impact of the bt2-H2328 mutation on these pathways and revealed that the bt2-H2328 mutation did not only affect the endosperm, but also the embryo at the metabolic level. These data suggest that, in the bt2-H2328 endosperms, regulatory networks are activated that redirect excess carbon into alternative biosynthetic pathways (amino acid synthesis) or into other tissues (embryo).

Fungal metabolism of toluene: monitoring of fluorinated analogs by (19)F nuclear magnetic resonance spectroscopy.

We used isomeric fluorotoluenes as model substrates to study the catabolism of toluene by five deuteromycete fungi and one ascomycete fungus capable of growth on toluene as the sole carbon and energy source, as well as by two fungi (Cunninghamella echinulata and Aspergillus niger) that cometabolize toluene. Whole cells were incubated with 2-, 3-, and 4-fluorotoluene, and metabolites were characterized by (19)F nuclear magnetic resonance. Oxidation of fluorotoluene by C. echinulata was initiated either at the aromatic ring, resulting in fluorinated o-cresol, or at the methyl group to form fluorobenzoate. The initial conversion of the fluorotoluenes by toluene-grown fungi occurred only at the side chain and resulted in fluorinated benzoates. The latter compounds were the substrate for the ring hydroxylation and, depending on the fluorine position, were further metabolized up to catecholic intermediates. From the (19)F nuclear magnetic resonance metabolic profiles, we propose that diverse fungi that grow on toluene assimilate toluene by an initial oxidation of the methyl group.

31P NMR studies of the diabetic lens.

Phosphorus-31 (31P) nuclear magnetic resonance (NMR) spectroscopic analyses of the crystalline lens from the experimental diabetic rat were performed. Qualitative and quantitative alterations in the phosphorus-31 NMR metabolic profile were observed over the course of 3 weeks after the induction of diabetes mellitus. Most striking was the appearance of two new, as yet unidentified, metabolites. These metabolites which resonate at 6.6 and 5.8 ppm were not detected in the normal lens. Compared to the normal lens, glycerol-3-phosphate (G3P) underwent an eightfold increase in concentration and phosphorylcholine decreased to one-third its initial level. The phosphodiesters, glycerophosphorylcholine (GPC) and glycerophosphorylethanolamine (GPE), decreased to barely detectable levels. Oral treatment of the diabetic animal with an aldose reductase inhibitor resulted in the preservation of an essentially normal lens 31P NMR spectrum. Except for the changes observed in glycerol-3-phosphate, these alterations have not been previously reported and raise new questions about the metabolic consequences of diabetes mellitus and the dependence of these alterations on the action of a single enzyme, aldose reductase.