Separating extracellular from intracellular fecal metabolites exposes cross-feeding architecture in the gut: exploring metabolite partitioning and ecological associations

The gut microbiota forms a complex ecosystem through metabolic interdependence (cross-feeding). However, conventional fecal metabolomics typically quantifies only total metabolite pools, making it difficult to distinguish extracellular-enriched metabolite pools from predominantly cell-associated ones, and thus limiting reconstruction of in situ metabolic networks. Here, we quantified fecal metabolites in paired fractions from the same specimen: an undisrupted fraction representing the extracellular pool and a strongly bead-beaten fraction representing the total pool; the intracellular pool was defined as the difference between total and extracellular measurements. Stool samples from 63 healthy individuals were analyzed for short-chain fatty acids, polyamines, and water-soluble vitamins, and the results were integrated with shotgun metagenomic profiles of species composition and functional genes. Vitamins exhibited two distinct behaviors. Adenosylcobalamin (a vitamin B12 coenzyme) was detectable only after disruption, and together with thiamine (B1) and niacin (B3) was classified as an intracellular-retained type (Type 1). In contrast, biotin (B7) and pantothenate (B5) showed higher extracellular proportions (Type 2). Integrative analyses further indicated that Type 1 thiamine was negatively associated with Blautia, consistent with intensive microbial utilization, whereas Type 2 biotin was strongly positively associated with Alistipes, suggesting links to ecological niches shaped by luminal pH and fermentation modes (carbohydrate vs protein fermentation). Fraction-resolved quantification provides a practical operational framework to differentiate extracellular from cell-associated metabolite pools, helping reconcile metagenomic potential with metabolomic reality and enabling deeper inference of cross-feeding structure in the gut ecosystem.