Development of a Quantitative Metabolomic Approach to Study Clinical Human Fecal Water Metabolome Based on Trimethylsilylation Derivatization and GC/MS Analysis

Gao, Xianfu; Pujos‐Guillot, Estelle; Sébédio, Jean‐Louis

doi:10.1021/ac1006552

Cited by 131 publications

(127 citation statements)

References 35 publications

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“…Metabolic profiling of fecal samples has been carried out using gas chromatography-mass spectrometry (GC-MS), liquid chromatography-MS (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy 9,10,11 . Published studies so far have focused on developing various fecal extraction and sample preparation methods for metabolic profiling 12,13 .…”

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confidence: 99%

Optimized Sample Handling Strategy for Metabolic Profiling of Human Feces

Gratton¹,

Phetcharaburanin²,

Mullish³

et al. 2016

Anal. Chem.

151

133

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Fecal metabolites are being increasingly studied to unravel the host-gut microbial metabolic interactions. However, there are currently no guidelines for fecal sample collection and storage based on a systematic evaluation of the effect of time, storage temperature, storage duration and sampling strategy. Here we derive an optimized protocol for fecal sample handling with the aim of maximizing metabolic stability and minimizing sample degradation. Samples obtained from five healthy individuals were analyzed to assess topographical homogeneity of feces, and to evaluate storage duration-, temperature-and freeze-thaw cycle-induced metabolic changes in crude stool and fecal water using a 1 H NMR spectroscopy-based metabolic profiling approach. Inter-individual variation was much greater than that attributable to storage conditions. Individual stool samples were found to be heterogeneous and spot sampling resulted in a high degree of metabolic variation. Crude fecal samples were remarkably unstable over time and exhibited distinct metabolic profiles at different storage temperatures. Microbial fermentation was the dominant driver in time-related changes observed in fecal samples stored at room temperature and this fermentative process was reduced when stored at 4°C. Crude fecal samples frozen at -20°C manifested elevated amino acids and nicotinate and depleted short chain fatty acids compared to crude fecal control samples. The relative concentrations of branched-chain and aromatic amino acids significantly increased in the freeze-thawed crude fecal samples, suggesting a release of microbial intracellular contents. The metabolic profiles of fecal water samples were more stable compared to crude samples. Our recommendation is that intact fecal samples should be collected, kept at 4°C or on ice during transportation, and extracted ideally within 1 h of collection, or a maximum of 24 h. Fecal water samples should be extracted from a representative amount (~15 g) of homogenized stool sample, aliquoted and stored at < -20°C, avoiding further freeze-thaw cycles.Metabolic profiling of biofluids and tissues generates data on a wide range of metabolites and provides extensive metabolic information on multiple biological processes in complex superorganisms such as mammals. Although urine and blood are often used to investigate systemic responses of animals and humans to various environmental stimuli or therapeutic interventions 1 , the search for disease biomarkers in fecal samples and studies on host-microbial interactions have intensified over the last decade. The human intestinal tract harbors >100 trillion microbial cells 2 and these microbes exert their influences on the human host primarily by metabolic signaling and therefore optimized methodologies for the study of microbial metabolic footprint is crucial to this field. Mounting evidence shows that the microbial composition and its collective metabolic activity profoundly impacts host physiology and modulates the disease risk of the host 3 . To investigate th...

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mentioning

confidence: 99%

Optimized Sample Handling Strategy for Metabolic Profiling of Human Feces

Gratton¹,

Phetcharaburanin²,

Mullish³

et al. 2016

Anal. Chem.

151

133

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“…Derivatization refers to the pretreatment process, which can change thermolabile substances and nonvolatile compounds into volatile and thermostable compounds by means of chemical approach. The common derivatization methods include the silylation reaction [26,29], alkylation reaction [30], and ethyl chloroformate reaction [31][32][33].…”

Section: Characteristics Of Gas Chromatography-mass Spectrometrymentioning

confidence: 99%

Novel methodologies in analysis of small molecule biomarkers and living cells

Chen

Zhu

2014

Tumor Biol.

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Enzyme-linked immuno-sorbent assay (ELISA) is widely used for biomarker detection. A good biomarker can distinguish patients from healthy or benign diseases. However, the ELISA method is not suitable for small molecule or trace substance detection. Along with the development of new technologies, an increasing level of biomaterials, especially small molecules, will be identified as novel biomarkers. Quantitative immuno-PCR, chromatography-mass spectrometry, and nucleic acid aptamer are emerging methodologies for detection of small molecule biomarkers, even in living cells. In this review, we focus on these novel technologies and their potential for small molecule biomarkers and living cell analysis.

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“…High-throughput analysis should be global, quantitative, robust, reproducible, accurate and interpretable. Hyphenated Gas Chromatography-Mass Spectrometry (GC-MS) is an analytical method that in recent years has evolved to enable simultaneous analysis of many different metabolites in urine samples due to its high resolution and high sensitivity [17,18]. Additionally, biological processes (such as metabolic response) are dynamic by nature and have temporal progressions and dynamic sampling will improve the detection of changes that occur over time following treatment.…”

Section: Introductionmentioning

confidence: 99%

Rats’ Metabolic Responses to Chronic Intervention with Cantonese Herbal Tea

Li¹

2016

Metabolomics

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Cantonese Herbal Tea (CHT) is consumed in south China to alleviate feelings of discomfort due to the over-heat and humidity in the body, but the related molecular mechanisms behind its effects remain obscure. The urinary metabolic responses to CHT over time in rats were investigated during a 38-day-period using GC-MS-based metabonomics. Integrated with student's t-test and Orthogonal Projection on Latent Structures Discriminant Analysis (OPLS-DA) at each time point revealed that the process could be divided into two stages. In the first stage, the bioefficacy was characterized by elevated antioxidant activity and the depletion of oxidative stresses. However, the effect was reduced with time and replaced by a second stage characterized by the decreased flow of energy metabolism and an imbalance in gut microbiota. In conclusion, metabonomics study gained insight into the metabolic changes induced by CHT intake and the extent to which CHT intake influences the urinary metabolome, which also provides a powerful method for understanding the mechanism of CHT.

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Development of a Quantitative Metabolomic Approach to Study Clinical Human Fecal Water Metabolome Based on Trimethylsilylation Derivatization and GC/MS Analysis

Cited by 131 publications

References 35 publications

Optimized Sample Handling Strategy for Metabolic Profiling of Human Feces

Optimized Sample Handling Strategy for Metabolic Profiling of Human Feces

Novel methodologies in analysis of small molecule biomarkers and living cells

Rats’ Metabolic Responses to Chronic Intervention with Cantonese Herbal Tea

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