**1. Introduction**

*Blastocystis* is a genus of anaerobic protozoan that resides in the gastrointestinal tract of many vertebrate species and has historically been classified as a parasite, yet its pathogenicity has been a subject of dispute in recent years. *Blastocystis* has a unique metabolism and possesses a mitochondrial-related organelle (MRO) with chimeric characteristics of an aerobic mitochondrion and hydrogenosomes [1]. Many of these characteristics have been acquired by lateral gene transfer from prokaryotes and possibly other eukaryotic organisms in the gastrointestinal tract, and these have likely supported the adaptation of *Blastocystis* to the gut environment [2].

Previous in vitro studies aimed at mapping the unique metabolic pathways in *Blastocystis* have been based on genome and transcriptome analyses [3–5]. Biochemical analysis has involved fractionation, the separation of organelles by isopycnic density and the analysis of absorbance following the addition of certain substrates [6]. The latter of these approaches monitors enzyme activity in different organelles based on available nutrients and added substrates in vitro. This approach is limited in the range of enzymes and pathways that can be monitored. Therefore, a technique in which the whole metabolome can be analysed in the context of the host or in vitro culture is required. Metabolomics is a technique which can be utilised to analyse the metabolome of a cell or microorganism. This technique has been used to analyse the metabolomes of many microbes [7,8], plants [9],

**Citation:** Newton, J.M.; Betts, E.L.; Yiangou, L.; Ortega Roldan, J.; Tsaousis, A.D.; Thompson, G.S. Establishing a Metabolite Extraction Method to Study the Metabolome of *Blastocystis* Using NMR. *Molecules* **2021**, *26*, 3285. https://doi.org/ 10.3390/molecules26113285

Academic Editor: Robert Brinson

Received: 30 April 2021 Accepted: 26 May 2021 Published: 29 May 2021

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nematodes [10] and animal cells [11–13]. Additionally, it has also been used to detect the molecules present in biological liquids such as blood [14], urine [14–16] and breast milk [17]. Mass spectrometry (MS) is probably the most popular analysis method for the detection and characterisation of small molecules and has been extremely successful because of its high sensitivity [10,18]. However, its arduous sample preparation can involve many steps to produce samples with good ionisation and MS properties. Subsequently, this can result in a loss of sample and the integrity of the metabolites being prejudiced. Therefore, reproducibility and accurate quantification can be difficult to achieve. In contrast, NMR can provide a simpler, more reproducible method for quantitative molecule detection, albeit with considerably lower sensitivity. NMR does not require the same laborious sample preparation that MS does, and the sample can remain intact throughout the analysis, thus making it a better quantitative tool [19–24]. However, for reasons of practicality and health and safety, NMR methods still require the extraction of metabolites from semi-solid samples such as cell cultures, as high resolution 1H NMR is a solution state method. The question then becomes which solvent and method should be used to best isolate the desired group of molecules from a sample. For example, methanol is commonly used to extract polar molecules [10,11,13], while chloroform is commonly used to extract non-polar molecules [10,11].

Currently, the only protozoan parasite to have its metabolome analysed by NMR is *Giardia lamblia* [7]. In this study, the metabolome of *G. lamblia* was analysed by high resolution 1H magic angle spinning (HR-MAS) NMR. HR-MAS does not require an extraction solvent as the cells remain intact [7]. However, HR-MAS experiments have some major drawbacks: firstly, they require a relaxation filter to exclude larger molecules such as proteins, as these produce a background unfavourable for the integration of sharper peaks, thus hampering quantification and comparison. [25]. The presence of this relaxation filter affects the sensitivity of the experiment and reduces the number of metabolites that can be detected. Secondly, HR-MAS experiments are limited by the volumes and quantities of samples that can be run with a maximum of 50 μL, which is at least ten times lower than the volumes usually used in liquid state NMR.

1H NMR spectra have a proven track record for metabolite analysis from a number of biofluids and extraction methods [10–15,17]. Therefore, a combination of 1H NMR metabolomics using a 1D-1H-NOESY pulse sequence with an extraction protocol that only extracts small molecules provides an effective method for mapping *Blastocystis* metabolic pathways.

Herein, we aimed to investigate different extraction approaches in order to develop the optimum step-by-step method to extract metabolites from *Blastocystis* for analysis via 1H NMR in order to analyse its metabolism.
