Title: An overview of fecal preparation for global metabolic profiling
Author: O. Deda H.G. Gika I.D. Wilson G.A. Theodoridis
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), http://dx.doi.org/10.1016/j.jpba.2015.02.006
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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: email@example.com
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.
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” . 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 . 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 . 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 .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 . 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 . The gut microflora can be categorized in four 26 major phyla: Firmicutes and Actinobacteria (Gram-positive) and Bacteroidetes and Proteobacteria (Gram-27