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

Abstract

Background The gut metabolome may be associated with the incidence and progression of numerous diseases. The composition of the gut metabolome can be captured by measuring metabolite levels in the feces. However, there is little data describing the effect of fecal sample collection methods on metabolomic measures. Methods We collected fecal samples from eighteen volunteers using four methods: no solution, 95% ethanol, fecal occult blood test (FOBT) cards, and fecal immunochemical test (FIT). One set of samples was frozen after collection (day 0), and for 95% ethanol, FOBT, and FIT, a second set was frozen after 96 hours at room temperature. We evaluated 1) technical reproducibility within sample replicates, 2) stability after 96 hours at room temperature for 95% ethanol, FOBT, and FIT, and 3) concordance of metabolite measures with the putative “gold standard,” day 0 samples without solution. Results Intraclass correlation coefficients (ICC) estimating technical reproducibility were high for replicate samples for each collection method. ICCs estimating stability at room temperature were high for 95% ethanol and FOBT (median ICC>0.87) but not FIT (median ICC=0.52). Similarly, Spearman correlation coefficients (rs) estimating metabolite concordance with the “gold standard” were higher for 95% ethanol (median rs=0.82) and FOBT (median rs=0.70) than for FIT (median rs=0.40). Conclusions Metabolomic measurements appear reproducible and stable in fecal samples collected with 95% ethanol or FOBT. Concordance with the “gold standard” is highest with 95% ethanol and acceptable with FOBT. Impact Future epidemiologic studies should collect feces using 95% ethanol or FOBT if interested in studying fecal metabolomics.

Reproducibility of assessing fecal microbiota in chronic constipation

“…Similar approaches have also been used to study the reproducibility and stability of various fecal sampling methods. [35][36][37][38][39] Overall, the agreement between two fecal samples for all measures of alpha and beta diversity in healthy people and patients with 57 (0.28-0.86) 0.89 (0.81-0 which, to speculate, may be more pronounced in people with normal compared to slow colonic transit. The fecal microbiota, stool consistency, and colonic transit are known to be univariately associated with each other.…”

“…The reason we chose the ICC measure over other measures is that ICC is directly related to statistical power, a key issue in the study design. Similar approaches have also been used to study the reproducibility and stability of various fecal sampling methods …”

Nutrimetabolomics: An Integrative Action for Metabolomic Analyses in Human Nutritional Studies

“…The easiest method for fecal storage is homogenization of the fresh stool sample by stirring with a sterile spatula directly in the delivery bag and then aliquoting a few milligrams to a few grams into feces tubes (i.e., Sarstedt) and storing at −80 °C . Le Galle et al .…”

“…Prestorage treatment and homogenization of fecal material is frequently done directly in ice‐cold phosphate‐buffered saline (PBS) by mixing representative volumes of feces with 2 volumes of PBS buffer . It has been shown that the addition of 95% ethanol, water/methanol, or twice distilled water tends to improve the overall recovery of fecal metabolites . Such prepared fecal slurry (fecal water) should be vortexed and ultracentrifuged at 35 000 × g for 2 h at 4 °C and the supernatant filtered, aliquoted, and stored at −80 °C.…”

ESI‐MS/MS analysis of protonated N‐methyl amino acids and their immonium ions

“…12 N-methyl Alanine was identified in human fecal sample. 13 In the current era of omics studies, N-methylated amino acids have been reported as potential biomarkers for several diseases like prostate cancer, chronic kidney disease (CKD), and cardiovascular disease.…”