Optimized Sample Handling Strategy for Metabolic Profiling of Human Feces

Abstract

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...

Effects of Sampling Conditions and Environmental Factors on Fecal Volatile Organic Compound Analysis by an Electronic Nose Device

“…Similar results were obtained in a study about fecal metabolite preservation [39]. Fecal samples were found to be highly unstable over time when left at room temperature, mainly due to ongoing fermentative processes, resulting in distinct metabolic profiles based on time-dependent differences between samples [39].…”

“…High temperatures lead to release of more different types of volatiles at increased concentrations compared to low temperatures, consequently influencing VOC composition [39,42]. This is probably the reason for the observed differences in VOC profiles measured at different temperatures in the present study.…”

“…Probably, production of metabolic co-products by intestinal microbes are halted during immediate freezing and do not recover completely upon thawing. However, recent fecal metabolomics studies demonstrated a significant increase in relative concentration of several branched-chain and aromatic amino acids in samples enduring one or multiple freeze–thaw cycles (both −20 °C and −80 °C) compared to fresh samples, suggesting a release of microbial intracellular contents following freeze–thaw cycles [27,39]. Notably, using GC-MS, similar fecal VOC profiles were detected deriving from fecal samples stored at −20 °C for seven days, compared to fresh samples, although, unfortunately, additional data was not published [40].…”

“…With higher sample temperature, more organic compounds become volatile with higher intensities. An additional explanation might be that microbial fermentation processes are increased at higher temperatures [39]. Since different VOCs have specific optimal temperatures to be in volatile state, optimal temperature depends on VOC of interest, which may differ for different (clinical) applications.…”

Comparison of Collection Methods for Fecal Samples for Discovery Metabolomics in Epidemiologic Studies

“…A number of other small case-control (34, 35), cross-sectional (36, 37), and intervention (38–40) studies have used NMR spectroscopy to generate metabolic phenotypes of various types of fecal samples, including crude fecal samples (37), fecal water extracts (34–37, 40) and lyophilized feces (38, 39). Although these studies have shown reasonable reproducibility, they have been more targeted, capturing a smaller less diverse set of metabolites than untargeted approaches, and collectively have not used standardized sample collection, storage, or preparation methods.…”