Reproducibility and Stability of Aqueous Metabolite Levels in Extracted Serum by NMR Spectroscopy

Miele, Matthew M.; Irving, Brian A.; Wenrich, Broc R.; Martin, Philliip L; Rovnyak, David

doi:10.2174/2213235x04666160711160340

Cited by 6 publications

(6 citation statements)

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“…Acetonitrile solubilizes aqueous metabolites, precipitates proteins, and strips viral membranes to promote subsequent safe sample handling. A number of solvent systems are employed for extracting metabolites from sera [32,[57][58][59][60], where the use of acetonitrile yields high metabolite recoveries, and methanol protocols may offer even more recovery [32]. The 1:2 (serum:ACN) ratio employed here yields high levels of metabolites with a strong reduction in macromolecular constituents, and also was shown to have linear recovery and reproduce ground truth for selected metabolites in mock samples [60].…”

Section: Organic Extraction Of Aqueous Metabolitesmentioning

confidence: 94%

“…A number of solvent systems are employed for extracting metabolites from sera [32,[57][58][59][60], where the use of acetonitrile yields high metabolite recoveries, and methanol protocols may offer even more recovery [32]. The 1:2 (serum:ACN) ratio employed here yields high levels of metabolites with a strong reduction in macromolecular constituents, and also was shown to have linear recovery and reproduce ground truth for selected metabolites in mock samples [60]. Samples were always on ice or refrigerated during handling, with one exception noted below.…”

Section: Organic Extraction Of Aqueous Metabolitesmentioning

confidence: 99%

“…There is an approx. 2 hr window from resuspension in which to measure spectra [60], so each sample was acquired for 1 hr (typical pw 90 = 6.5 µs; typical DSS linewidth = 0.90 Hz; 256 scans; 8 steady state scans; 100 ms NOESY period); each scan consumed 15 s (4 s acquisition; 11 s recycle time). The presat period was 2 s (included in 11 s recycle time).…”

Section: Nmr Data Acquisitionmentioning

confidence: 99%

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Aqueous Metabolite Trends for the Progression of Nonalcoholic Fatty Liver Disease in Female Bariatric Surgery Patients by Targeted 1H-NMR Metabolomics

Robinson

Taddeo

Chu³

et al. 2021

Metabolites

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Determining biomarkers and better characterizing the biochemical progression of nonalcoholic fatty liver disease (NAFLD) remains a clinical challenge. A targeted 1H-NMR study of serum, combined with clinical variables, detected and localized biomarkers to stages of NAFLD in morbidly obese females. Pre-surgery serum samples from 100 middle-aged, morbidly obese female subjects, grouped on gold-standard liver wedge biopsies (non-NAFLD; steatosis; and fibrosis) were collected, extracted, and analyzed in aqueous (D2O) buffer (1H, 600 MHz). Profiled concentrations were subjected to exploratory statistical analysis. Metabolites varying significantly between the non-NAFLD and steatosis groups included the ketone bodies 3-hydroxybutyrate (↓; p = 0.035) and acetone (↓; p = 0.012), and also alanine (↑; p = 0.004) and a putative pyruvate signal (↑; p = 0.003). In contrast, the steatosis and fibrosis groups were characterized by 2-hydroxyisovalerate (↑; p = 0.023), betaine (↓; p = 0.008), hypoxanthine (↓; p = 0.003), taurine (↓; p = 0.001), 2-hydroxybutyrate (↑; p = 0.045), 3-hydroxyisobutyrate (↑; p = 0.046), and increasing medium chain fatty acids. Exploratory classification models with and without clinical variables exhibited overall success rates ca. 75–85%. In the study conditions, inhibition of fatty acid oxidation and disruption of the hepatic urea cycle are supported as early features of NAFLD that continue in fibrosis. In fibrosis, markers support inflammation, hepatocyte damage, and decreased liver function. Complementarity of NMR concentrations and clinical information in classification models is shown. A broader hypothesis that standard-of-care sera can yield metabolomic information is supported.

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Section: Organic Extraction Of Aqueous Metabolitesmentioning

confidence: 94%

Section: Organic Extraction Of Aqueous Metabolitesmentioning

confidence: 99%

Section: Nmr Data Acquisitionmentioning

confidence: 99%

See 1 more Smart Citation

Aqueous Metabolite Trends for the Progression of Nonalcoholic Fatty Liver Disease in Female Bariatric Surgery Patients by Targeted 1H-NMR Metabolomics

Robinson

Taddeo

Chu³

et al. 2021

Metabolites

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“…The chloroform:methanol method, however, involves significant sample handling and lower metabolite recovery. Approaches involving extractions with other solvents have been proposed for greater recovery of metabolites [27][28][29]. This work focused particularly on developing optimal acetonitrile extractions of individual bee samples.…”

Section: Extraction Methodologymentioning

confidence: 99%

Methodology for Single Bee and Bee Brain 1H-NMR Metabolomics

et al. 2021

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The feasibility of metabolomic 1H NMR spectroscopy is demonstrated for its potential to help unravel the complex factors that are impacting honeybee health and behavior. Targeted and non-targeted 1H NMR metabolic profiles of liquid and tissue samples of organisms could provide information on the pathology of infections and on environmentally induced stresses. This work reports on establishing extraction methods for NMR metabolic characterization of Apis mellifera, the European honeybee, describes the currently assignable aqueous metabolome, and gives examples of diverse samples (brain, head, body, whole bee) and biologically meaningful metabolic variation (drone, forager, day old, deformed wing virus). Both high-field (600 MHz) and low-field (80 MHz) methods are applicable, and 1H NMR can observe a useful subset of the metabolome of single bees using accessible NMR instrumentation (600 MHz, inverse room temperature probe) in order to avoid pooling several bees. Metabolite levels and changes can be measured by NMR in the bee brain, where dysregulation of metabolic processes has been implicated in colony collapse. For a targeted study, the ability to recover 10-hydroxy-2-decenoic acid in mandibular glands is shown, as well as markers of interest in the bee brain such as GABA (4-aminobutyrate), proline, and arginine. The findings here support the growing use of 1H NMR more broadly in bees, native pollinators, and insects.

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“…PCA and PLS-DA reduces this information to a single point on a 2D or 3D score plot, based on the influence of particular variables in which maximum variance between samples is attributed. These clustering methods allow simple evaluation of sample-to-sample variation and, thus, the interpretation of reproducibility and class differences [13]. Experimental parameters should be identical amongst samples to obtain reliable data [7].…”

Section: Introductionmentioning

confidence: 99%

Influence of Metabolite Extraction Methods on 1H-NMR-Based Metabolomic Profiling of Enteropathogenic Yersinia

Gines

Collier

Abdalla

et al. 2018

MPs

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Metabolite extraction is one of the critical steps in microbial metabolome analysis. It affects both the observed metabolite content and biological interpretation of the data. Several methods exist for metabolite extraction of microbes, but the literature is not consistent regarding the sample model, adequacy, and performance of each method. In this study, an optimal extraction protocol for Yersinia intracellular metabolites was investigated. The effect of five extraction protocols consisting of different extraction solvent systems (60% methanol, 100% methanol, acetonitrile/methanol/water (2:2:1), chloroform/methanol/water (2:1:1), and 60% ethanol) on Yersinia metabolic profiles were compared. The number of detected peaks, sample-to-sample variation, and metabolite yield were used as criteria. Extracted metabolites were analyzed by 1H-NMR and principal component analysis (PCA), as well as partial least squares discriminant analysis (PLS-DA) multivariate statistics. The extraction protocol using 100% methanol as the extraction solvent provided the highest number of detected peaks for both Yersinia species analyzed, yielding more spectral information. Together with the reproducibility and spectrum quality, 100% methanol extraction was suitable for intracellular metabolite extraction from both species. However, depending on the metabolites of interest, other solvents might be more suitable for future studies, as distinct profiles were observed amongst the extraction methods.

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Reproducibility and Stability of Aqueous Metabolite Levels in Extracted Serum by NMR Spectroscopy

Cited by 6 publications

References 0 publications

Aqueous Metabolite Trends for the Progression of Nonalcoholic Fatty Liver Disease in Female Bariatric Surgery Patients by Targeted 1H-NMR Metabolomics

Aqueous Metabolite Trends for the Progression of Nonalcoholic Fatty Liver Disease in Female Bariatric Surgery Patients by Targeted 1H-NMR Metabolomics

Methodology for Single Bee and Bee Brain 1H-NMR Metabolomics

Influence of Metabolite Extraction Methods on 1H-NMR-Based Metabolomic Profiling of Enteropathogenic Yersinia

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