An overview of fecal sample preparation for global metabolic profilingby Olga Deda, Helen G. Gika, Ian D. Wilson, Georgios A. Theodoridis

Journal of Pharmaceutical and Biomedical Analysis


Analytical Chemistry / Spectroscopy / Drug Discovery / Pharmaceutical Science / Clinical Biochemistry


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Marie Joossens, Geert Huys, Kristel Van Steen, Margo Cnockaert, Séverine Vermeire, Paul Rutgeerts, Kristin Verbeke, Peter Vandamme, Vicky De Preter


Accepted Manuscript

Title: An overview of fecal preparation for global metabolic profiling

Author: O. Deda H.G. Gika I.D. Wilson G.A. Theodoridis

PII: S0731-7085(15)00101-6


Reference: PBA 9947

To appear in: Journal of Pharmaceutical and Biomedical Analysis

Received date: 3-2-2015

Accepted date: 5-2-2015

Please cite this article as: O. Deda, H.G. Gika, I.D. Wilson, G.A. Theodoridis, An overview of fecal preparation for global metabolic profiling, Journal of Pharmaceutical and Biomedical Analysis (2015),

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Page 1 of 35

Ac ce pte d M an us cri pt 1

An overview of fecal preparation for global metabolic profiling

O. Dedaa, H. G. Gikab*, I. D. Wilsonc G. A. Theodoridisa a Department of Chemistry, Aristotle University of Thessaloniki, 54124 Greece b Department of Chemical Engineering, Aristotle University, Thessaloniki 54124, Greece c Dept. of Surgery and Cancer, Imperial College, Exhibition Rd, South Kensington, London, SW7 2AZ, UK *Author for correspondence:

Tel: +302310996224

HIGHLIGHTS  Review on the sample preparation of feces for metabolomics analysis  Extraction, filtration and other preparation procedures are discussed  Feces provide valuable information for biomarker discovery  Major application field in health and nutrition studies  host-guest interactions (gut microflora) can be elicited

Page 2 of 35

Ac ce pte d M an us cri pt 2


The global metabolic profiling of feces represents a challenge for both analytical chemistry and biochemistry standpoints. As a specimen, feces is complex, not homogenous and rich in macromolecules and particulate, non-digested, matter that can present problems for analytical systems. Further to this, the composition of feces is highly dependent on short-term dietary factors whilst also representing the primary specimen where co-metabolism of the host organism and the gut-microbiota is expressed. Thus the presence and the content of metabolites can be a result of host metabolism, gut microbiota metabolism or co-metabolism. Successful sample preparation and metabolite analysis require that the methodology applied for sample preparation is adequate to compensate for the highly variable nature of the sample in order to generate useful data and provide insight to ongoing biochemical processes, thereby generating hypotheses. The current practice for processing fecal samples for global metabolic profiling are described with emphasis on critical aspects in sample preparation: e.g., homogenization, filtration, centrifugation, solvent extraction and so forth are presented and conditions/parameter selection are discussed. The different methods applied for sample preparation of feces prior to metabolite analysis are summarized and illustrated using selected examples to highlight the effect of sample preparation on the metabolic profile obtained.

Key words

Feces, Metabolomics, Metabonomics, Metabolic profiling, disease biomarker discovery, gut microflora

Page 3 of 35

Ac ce pte d M an us cri pt 3 1.Introduction1

Global metabolic profiling, known by the terms metabolomics and metabonomics, is being increasing 2 employed to determine metabolic phenotypes, derive novel biomarkers of various conditions and generate 3 hypotheses. Metabolomics is defined in the Oxford English dictionary as “the scientific study of the set 4 of metabolites present within an organism, cell, or tissue” whilst metabonomics is “the quantitative 5 measurement of the dynamic multi parametric metabolic response of living systems to physiological stimuli 6 or genetic modification” [1]. This simultaneous detection, identification and quantification of a large 7 number of metabolites, often at extremely varied concentrations, is technically very demanding so each 8 link of this chain must be studied and optimized. The most widely used analytical platforms are nuclear 9 magnetic resonance (NMR) spectroscopy, gas chromatography mass spectrometry (GC-MS) and liquid 10 chromatography mass spectrometry (LC-MS) [2-4].11

Numerous studies reflect the need to improve the detection, the identification and particularly the data 12 processing, in order to derive reliable data from metabolic profiling studies [5-11]. One could argue that the 13 process of the sample preparation prior to metabolomics analysis has not received the necessary attention14 [12]. As a result there is no universal approach in the preparation of samples for holistic analysis. Limiting 15 factors are the matrix diversity and also sample physiological variation [13]. There is arguably significant 16 need for the development of stable, repeatable methods for sample preparation. An optimal sample 17 preparation should be short, reproducible, capable of extracting all metabolites while avoiding sample 18 alteration through the process [14].19

In life sciences the specimens of interest are biofluids such as plasma, serum, urine bile etc., and tissue. An 20 interesting specimen which has not been in the center of research in literature is feces. In fact, stool 21 material is the most appropriate biospecimen in order to reveal the effect of gut microbiota in host 22 metabolism. Trillions of microbiota coexist in the human or animal intestine creating a human-microbe 23 hybrid called super-organism [15]. The symbiotic gut microbiota interacts with the host and affects the 24 metabolism creating co-metabolism. The population of the hundreds species of bacteria plays a significant 25 role, affecting the balance between health and disease [16]. The gut microflora can be categorized in four 26 major phyla: Firmicutes and Actinobacteria (Gram-positive) and Bacteroidetes and Proteobacteria (Gram-27