36 research outputs found
NMR Spectroscopy of Cell Culture, Tissues, and Other Biofluids
NMR spectroscopy can provide a wealth of information on cellular metabolism and is frequently used in metabolomics application that use cultured cells, tissues, and whole organisms. Central to these analyses are the protocols for sample harvest, which incorporate procedures for quenching metabolic processes to preserve samples in a state that is representative of their source. In this chapter, the main considerations are discussed with reference to literature exemplars. In the latter half of the chapter, less commonly studied biofluids that also have specific sample preparation requirements are discussed, with a focus on cerebrospinal fluid, faeces, bile, seminal fluid, and milk.</jats:p
Oxidative stress and inflammation induced by environmental and psychological stressors: a biomarker perspective
Significance. The environment can elicit biological responses such as oxidative stress (OS) and inflammation as consequence of chemical, physical or psychological changes. As population studies are essential for establishing these environment-organism interactions, biomarkers of oxidative stress or inflammation are critical in formulating mechanistic hypotheses. Recent advances. By using examples of stress induced by various mechanisms, we focus on the biomarkers that have been used to assess oxidative stress and inflammation in these conditions. We discuss the difference between biomarkers that are the result of a chemical reaction (such as lipid peroxides or oxidized proteins that are a result of the reaction of molecules with reactive oxygen species, ROS) and those that represent the biological response to stress, such as the transcription factor NRF2 or inflammation and inflammatory cytokines. Critical issues. The high-throughput and holistic approaches to biomarker discovery used extensively in large-scale molecular epidemiological exposome are also discussed in the context of human exposure to environmental stressors. Future directions. We propose to consider the role of biomarkers as signs and distinguish between signs that are just indicators of biological processes and proxies that one can interact with and modify the disease process
Assessment of metabolic phenotypic variability in children's urine using 1H NMR spectroscopy
The application of metabolic phenotyping in clinical and
epidemiological studies is limited by a poor understanding of
inter-individual, intra-individual and temporal variability in
metabolic phenotypes. Using 1H NMR spectroscopy we characterised
short-term variability in urinary metabolites measured from 20
children aged 8-9 years old. Daily spot morning, night-time and
pooled (50:50 morning and night-time) urine samples across six
days (18 samples per child) were analysed, and 44 metabolites
quantified. Intraclass correlation coefficients (ICC) and mixed
effect models were applied to assess the reproducibility and
biological variance of metabolic phenotypes. Excellent
analytical reproducibility and precision was demonstrated for
the 1H NMR spectroscopic platform (median CV 7.2%). Pooled
samples captured the best inter-individual variability with an
ICC of 0.40 (median). Trimethylamine, N-acetyl neuraminic acid,
3-hydroxyisobutyrate, 3-hydroxybutyrate/3-aminoisobutyrate,
tyrosine, valine and 3-hydroxyisovalerate exhibited the highest
stability with over 50% of variance specific to the child. The
pooled sample was shown to capture the most inter-individual
variance in the metabolic phenotype, which is of importance for
molecular epidemiology study design. A substantial proportion of
the variation in the urinary metabolome of children is specific
to the individual, underlining the potential of such data to
inform clinical and exposome studies conducted early in life
Kinetic modelling of acyl glucuronide and glucoside reactivity and development of structure-property relationships.
Acyl glucuronide metabolites have been implicated in the toxicity of several carboxylic acid-containing drugs, and the rate of their degradation via intramolecular transacylation and hydrolysis has been associated with the degree of protein adduct formation. Although not yet proven, the formation of protein adducts in vivo - and subsequent downstream effects - has been proposed as a mechanism of toxicity for carboxylic acid-containing xenobiotics capable of forming acyl glucuronides. A structurally-related series of metabolites, the acyl glucosides, have also been shown to undergo similar degradation reactions and consequently the potential to display a similar mode of toxicity. Here we report detailed kinetic models of each transacylation and hydrolysis reaction for a series of phenylacetic acid acyl glucuronides and their analogous acyl glucosides. Differences in reactivity were observed for the individual transacylation steps between the compound series; our findings suggest that the charged carboxylate ion and neutral hydroxyl group in the glucuronide and glucoside conjugates, respectively, are responsible for these differences. The transacylation reaction was modelled using density functional theory and the calculated activation energy for this reaction showed a close correlation with the degradation rate of the 1-β anomer. Comparison of optimised geometries between the two series of conjugates revealed differences in hydrogen bonding which may further explain the differences in reactivity observed. Together, these models may find application in drug discovery for prediction of acyl glucuronide and glucoside metabolite behaviour
Consensus-Phenotype Integration of Transcriptomic and Metabolomic Data Implies a Role for Metabolism in the Chemosensitivity of Tumour Cells
Using transcriptomic and metabolomic measurements from the NCI60 cell line panel,
together with a novel approach to integration of molecular profile data, we show
that the biochemical pathways associated with tumour cell chemosensitivity to
platinum-based drugs are highly coincident, i.e. they describe a consensus
phenotype. Direct integration of metabolome and transcriptome data at the point
of pathway analysis improved the detection of consensus pathways by 76%,
and revealed associations between platinum sensitivity and several metabolic
pathways that were not visible from transcriptome analysis alone. These pathways
included the TCA cycle and pyruvate metabolism, lipoprotein uptake and
nucleotide synthesis by both salvage and de novo pathways. Extending the
approach across a wide panel of chemotherapeutics, we confirmed the specificity
of the metabolic pathway associations to platinum sensitivity. We conclude that
metabolic phenotyping could play a role in predicting response to platinum
chemotherapy and that consensus-phenotype integration of molecular profiling
data is a powerful and versatile tool for both biomarker discovery and for
exploring the complex relationships between biological pathways and drug
response
Determinants of the urinary and serum metabolome in children from six European populations
Background Environment and diet in early life can affect development and health throughout the life course. Metabolic phenotyping of urine and serum represents a complementary systems-wide approach to elucidate environment–health interactions. However, large-scale metabolome studies in children combining analyses of these biological fluids are lacking. Here, we sought to characterise the major determinants of the child metabolome and to define metabolite associations with age, sex, BMI and dietary habits in European children, by exploiting a unique biobank established as part of the Human Early-Life Exposome project (http://www.projecthelix.eu). Methods Metabolic phenotypes of matched urine and serum samples from 1192 children (aged 6–11) recruited from birth cohorts in six European countries were measured using high-throughput 1H nuclear magnetic resonance (NMR) spectroscopy and a targeted LC-MS/MS metabolomic assay (Biocrates AbsoluteIDQ p180 kit). Results We identified both urinary and serum creatinine to be positively associated with age. Metabolic associations to BMI z-score included a novel association with urinary 4-deoxyerythronic acid in addition to valine, serum carnitine, short-chain acylcarnitines (C3, C5), glutamate, BCAAs, lysophosphatidylcholines (lysoPC a C14:0, lysoPC a C16:1, lysoPC a C18:1, lysoPC a C18:2) and sphingolipids (SM C16:0, SM C16:1, SM C18:1). Dietary-metabolite associations included urinary creatine and serum phosphatidylcholines (4) with meat intake, serum phosphatidylcholines (12) with fish, urinary hippurate with vegetables, and urinary proline betaine and hippurate with fruit intake. Population-specific variance (age, sex, BMI, ethnicity, dietary and country of origin) was better captured in the serum than in the urine profile; these factors explained a median of 9.0% variance amongst serum metabolites versus a median of 5.1% amongst urinary metabolites. Metabolic pathway correlations were identified, and concentrations of corresponding metabolites were significantly correlated (r > 0.18) between urine and serum. Conclusions We have established a pan-European reference metabolome for urine and serum of healthy children and gathered critical resources not previously available for future investigations into the influence of the metabolome on child health. The six European cohort populations studied share common metabolic associations with age, sex, BMI z-score and main dietary habits. Furthermore, we have identified a novel metabolic association between threonine catabolism and BMI of children
Metabolic profiling detects early effects of environmental and lifestyle exposure to cadmium in a human population
Background: The ‘exposome’ represents the accumulation of all environmental exposures across a lifetime. Topdown
strategies are required to assess something this comprehensive, and could transform our understanding of
how environmental factors affect human health. Metabolic profiling (metabonomics/metabolomics) defines an
individual’s metabolic phenotype, which is influenced by genotype, diet, lifestyle, health and xenobiotic exposure,
and could also reveal intermediate biomarkers for disease risk that reflect adaptive response to exposure. We
investigated changes in metabolism in volunteers living near a point source of environmental pollution: a closed
zinc smelter with associated elevated levels of environmental cadmium. Methods: High-resolution 1H NMR spectroscopy (metabonomics) was used to acquire urinary metabolic profiles
from 178 human volunteers. The spectral data were subjected to multivariate and univariate analysis to identify
metabolites that were correlated with lifestyle or biological factors. Urinary levels of 8-oxo-deoxyguanosine were
also measured, using mass spectrometry, as a marker of systemic oxidative stress. Results: Six urinary metabolites, either associated with mitochondrial metabolism (citrate, 3-hydroxyisovalerate, 4-
deoxy-erythronic acid) or one-carbon metabolism (dimethylglycine, creatinine, creatine), were associated with
cadmium exposure. In particular, citrate levels retained a significant correlation to urinary cadmium and smoking
status after controlling for age and sex. Oxidative stress (as determined by urinary 8-oxo-deoxyguanosine levels)
was elevated in individuals with high cadmium exposure, supporting the hypothesis that heavy metal
accumulation was causing mitochondrial dysfunction. Conclusions: This study shows evidence that an NMR-based metabolic profiling study in an uncontrolled human
population is capable of identifying intermediate biomarkers of response to toxicants at true environmental
concentrations, paving the way for exposome research.
Keywords: metabonomics, cadmium, environmental health, exposome, metabolomics, molecular epidemiolog
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NMR in environmental and nutritional research
Small molecular species represent environmental/nutritional exposures as well as downstream mediators and their modulation reflects consequences of those exposures; metabolome analyses are therefore critical in efforts to characterise the internal chemical milieu to complement genomic profiles. As in other areas of health research, NMR spectroscopy is a primary platform for biofluid analysis, benefitting from good reproducibility and robustness, wide metabolome coverage, and the capacity to provide quantitative data. In this chapter, we highlight some of the main applications of NMR in environmental and nutritional research related to human health, which include analysis of dietary components, to molecular phenotyping, and structure elucidation of novel metabolites.</jats:p
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 [...