The Importance of Objective Stool Classification in Fecal 1H-NMR Metabolomics: Exponential Increase in Stool Crosslinking Is Mirrored in Systemic Inflammation and Associated to Fecal Acetate and Methionine

Abstract

Past studies strongly connected stool consistency-as measured by Bristol Stool Scale (BSS)-with microbial gene richness and intestinal inflammation, colonic transit time and metabolome characteristics that are of clinical relevance in numerous gastro intestinal conditions. While retention time, defecation rate, BSS but not water activity have been shown to account for BSS-associated inflammatory effects, the potential correlation with the strength of a gel in the context of intestinal forces, abrasion, mucus imprinting, fecal pore clogging remains unexplored as a shaping factor for intestinal inflammation and has yet to be determined. Our study introduced a minimal pressure approach (MP) by probe indentation as measure of stool material crosslinking in fecal samples. Results reported here were obtained from 170 samples collected in two independent projects, including males and females, covering a wide span of moisture contents and BSS. MP values increased exponentially with increasing consistency (i.e., lower BSS) and enabled stratification of samples exhibiting mixed BSS classes. A trade-off between lowest MP and highest dry matter content delineated the span of intermediate healthy density of gel crosslinks. The crossectional transects identified fecal surface layers with exceptionally high MP and of <5 mm thickness followed by internal structures with an order of magnitude lower MP, characteristic of healthy stool consistency. The MP and BSS values reported in this study were coupled to reanalysis of the PlanHab data and fecal 1H-NMR metabolomes reported before. The exponential association between stool consistency and MP determined in this study was mirrored in the elevated intestinal and also systemic inflammation and other detrimental physiological deconditioning effects observed in the PlanHab participants reported before. The MP approach described in this study can be used to better understand fecal hardness and its relationships to human health as it provides a simple, fine scale and objective stool classification approach for the characterization of the exact sampling locations in future microbiome and metabolome studies.

Keywords: BSS; Bristol stool scale; JADBIO; classification; feces; gel network; intestinal abrasion; stool.

Figure 1

(A) Large heterogeneity in stool surface minimal pressure (MP (g/3.14 mm²)) was identified within the same BSS class. Note the nonlinear increase in MP. (B) Healthy BSS values (3–4) were concentrated around the trade-off between the lowest MP and highest dry matter content. The intermediate density of crosslinks is beneficial for maintenance of human health as based on (•) 78 sample collection and our PlanHab project data (n = 96; [3,13,14]) (•) and (•)). (C) An example of stool containing mixed BSS classes (•) and more than an order of magnitude difference in surface minimal pressure (•) along the longitudinal transect of fecal specimen scanned from all 4 sides (n = 4). (D) The crossectional (lateral) transects of the same stool example as shown in (C) and their respective internal MP values at locations 3, 6, 9, 12 and 15 cm from the fecal tip. A decrease in surface MP and relatively small change in internal MP values can be seen along fecal specimen. Please consult Figures S4 and S5 for more details.

Figure 2

Presentation of MP values for the PlanHab samples in relation to the experimental variants described with BSS (Figure S6). Error bars designate standard deviation. NBR-normoxic bedrest, HBR-hypoxic bedrest, HAmb-hypoxic ambulatory variants of the PlanHab experiment.

Figure 3

An overview of the model performance in three different groups between fecal metabolomes in delineation with three different groups of MP (MP1 < 30 (A), 30 < MP2 < 75 (B) and MP3 > 75 (C)). Please note the rather uncharacteristic and hard to classify makeup of metabolomes observed in the intermediate, healthy, group (B). Horizontal and vertical dashed lines represent 95% confidence interval for false positive and true positive rates, respectively. The black dashed line represents model performance in case of random guessing; blue line represents training mean performance of the model.