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Establishing a Metabolite Extraction Method to Study the Metabolome of Blastocystis Using NMR.

ABSTRACT: Blastocystis is an opportunistic parasite commonly found in the intestines of humans and other animals. Despite its high prevalence, knowledge regarding Blastocystis biology within and outside the host is limited. Analysis of the metabolites produced by this anaerobe could provide insights that can help map its metabolism and determine its role in both health and disease. Due to its controversial pathogenicity, these metabolites could define its deterministic role in microbiome's "health" and/or subsequently resolve Blastocystis' potential impact in gastrointestinal health. A common method for elucidating the presence of these metabolites is through ¹H nuclear magnetic resonance (NMR). However, there are currently no described benchmarked methods available to extract metabolites from Blastocystis for ¹H NMR analysis. Herein, several extraction solvents, lysis methods and incubation temperatures were compared for their usefulness as an extraction protocol for this protozoan. Following extraction, the samples were freeze-dried, re-solubilized and analysed with ¹H NMR. The results demonstrate that carrying out the procedure at room temperature using methanol as an extraction solvent and bead bashing as a lysis technique provides a consistent, reproducible and efficient method to extract metabolites from Blastocystis for NMR.

SUBMITTER: Newton JM

PROVIDER: S-EPMC8199492 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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Establishing a Metabolite Extraction Method to Study the Metabolome of Blastocystis Using NMR.

Newton Jamie M JM Betts Emma L EL Yiangou Lyto L Ortega Roldan Jose J Tsaousis Anastasios D AD Thompson Gary S GS

Molecules (Basel, Switzerland) 20210529 11

Blastocystis is an opportunistic parasite commonly found in the intestines of humans and other animals. Despite its high prevalence, knowledge regarding Blastocystis biology within and outside the host is limited. Analysis of the metabolites produced by this anaerobe could provide insights that can help map its metabolism and determine its role in both health and disease. Due to its controversial pathogenicity, these metabolites could define its deterministic role in microbiome's " ...[more]

PMID: 34072445

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Project description:A current challenge in metabolomics is the reliable quantitation of many metabolites. Limited resolution and sensitivity combined with the challenges associated with unknown metabolite identification have restricted both the number and the quantitative accuracy of blood metabolites. Focused on alleviating this bottleneck in NMR-based metabolomics, investigations of pooled human serum combining an array of 1D/2D NMR experiments at 800 MHz, database searches, and spiking with authentic compounds enabled the identification of 67 blood metabolites. Many of these (?1/3) are new compared with those reported previously as a part of the Human Serum Metabolome Database. In addition, considering both the high reproducibility and quantitative nature of NMR as well as the sensitivity of NMR chemical shifts to altered sample conditions, experimental protocols and comprehensive peak annotations are provided here as a guide for identification and quantitation of the new pool of blood metabolites for routine applications. Further, investigations focused on the evaluation of quantitation using organic solvents revealed a surprisingly poor performance for protein precipitation using acetonitrile. One-third of the detected metabolites were attenuated by 10-67% compared with methanol precipitation at the same solvent-to-serum ratio of 2:1 (v/v). Nearly 2/3 of the metabolites were further attenuated by up to 65% upon increasing the acetonitrile-to-serum ratio to 4:1 (v/v). These results, combined with the newly established identity for many unknown metabolites in the NMR spectrum, offer new avenues for human serum/plasma-based metabolomics. Further, the ability to quantitatively evaluate nearly 70 blood metabolites that represent numerous classes, including amino acids, organic acids, carbohydrates, and heterocyclic compounds, using a simple and highly reproducible analytical method such as NMR may potentially guide the evaluation of samples for analysis using mass spectrometry.

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Establishing a Metabolite Extraction Method to Study the Metabolome of Blastocystis Using NMR.

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Establishing a Metabolite Extraction Method to Study the Metabolome of <i>Blastocystis</i> Using NMR.

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Establishing a Metabolite Extraction Method to Study the Metabolome of Blastocystis Using NMR.

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Establishing a Metabolite Extraction Method to Study the Metabolome of &lt;i&gt;Blastocystis&lt;/i&gt; Using NMR.

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OmicsDI is part of the ELIXIR infrastructure

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Establishing a Metabolite Extraction Method to Study the Metabolome of <i>Blastocystis</i> Using NMR.