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.

An Optimised Monophasic Faecal Extraction Method for LC-MS Analysis and its Application in Gastrointestinal Disease

“…This result is in agreement with a recently reported study, where MeOH was chosen as the optimal solvent for extraction of metabolites from human faecal samples to assess gut health [19]. Furthermore, MeOH has been found to be the optimal extraction solvent in a range of metabolomics studies including investigation of dietary influences in faecal samples [3], serum metabolite profiling [20], and Blastocystis ’ metabolism [21]. In comparison to one of the current most used extraction solvents, phosphate buffer saline (PBS) [22], the recognition of MeOH as an efficient organic buffer and resultant choice in a range of sample preparation methods may be attributed to effective protein denaturation [23] and multi-polarity chemical capture [24].…”

Metabolic Fluctuations in the Human Stool Obtained from Blastocystis Carriers and Non-Carriers

“…To our knowledge, this is the first report of a metabolomics-based study carried out on Blastocystis in humans. The focus was to assess metabolite profiles from Blastocystis carriers (B+) and non-carriers (B−) from three different countries and explore possible associations of the organism with the host stool metabolome by using 1 H NMR spectroscopy [34,39]. Findings revealed a distinct variation in the metabolome of positive carriers compared to non-carriers, with a number of distinguishable metabolites found between the groups.…”

“…While previous reports have documented the gut microbiome profiles of human hosts positively and negatively colonised with Blastocystis, similar investigations at the level of the metabolome are non-existent. 1D 1 H Nuclear Magnetic Resonance (NMR) is a quantitative and reproducible metabolite detection method, which has been previously used to analyse the metabolomes of many bacterial [32], mammalian [33,34], plant [35], fungal [36] and protozoan [37][38][39] cells/organisms. In recent years, there has been a marked increase in the use of metabolomics-based studies for exploring host parasite interactions [37,[40][41][42], as well as aiding research into chemotherapeutics [43,44].…”

The use of ecological analytical tools as an unconventional approach for untargeted metabolomics data analysis: the case of Cecropia obtusifolia and its adaptive responses to nitrate starvation

“…For more details, see Cadena-Zamudio et al ( 2020 ) and Monribot-Villanueva et al ( 2019 ). In this study, we use methanol as the extraction solvent because it has been commonly used to successfully extract polar molecules from diverse biological samples and, in some cases, it has allowed the detection of lower abundance metabolites, increasing the number of putative metabolites that can be identified (Borges et al 2020 ; Dunn et al 2011 ; Newton et al 2021 ; Saw et al 2021 ; Sitnikov et al 2016 ; Yang et al 2019 ).…”