Exploratory Metabolomic and Metagenomic Profiling of Fecal Samples in Isolated REM Sleep Behavior Disorder

Isolated REM sleep behavior disorder (iRBD) is among the most reliable prodromal markers of α-synucleinopathies. Under the body-first (gut-first) model of α-synuclein pathology, in which pathology may originate in the enteric nervous system and ascend to the brain, the gut is a biologically plausible compartment in which early and accessible molecular alterations might be detectable; however, the fecal metabolome remains poorly investigated in this population. In this exploratory, cross-sectional study of 21 iRBD patients and 15 healthy controls, we characterized fecal samples through an integrated multi-omics framework. The fecal metabolome was profiled on two complementary mass spectrometry platforms: HS-SPME GC-EI-high-resolution MS, used for both untargeted volatilomics and targeted quantification of linear and branched short-chain fatty acids, and untargeted LC-MS/MS for the non-volatile fraction. Gut microbial community composition was characterized on the same material by 16S rRNA gene sequencing, and the metabolomic and microbial layers were combined by DIABLO multi-omics integration. Linear short-chain fatty acids were preserved, whereas branched-chain fatty acids were selectively increased in iRBD, indicating a shift toward proteolytic fermentation. Volatile indole was reduced, while p-cresol and 2-octanone were increased, paralleled at the LC-MS/MS level by attenuation of the tryptophan–indole axis, class-level bile acid depletion, and increased acylcarnitines. The microbiome was depleted of Blautia and Faecalibacterium and enriched in Oscillospirales. Integration resolved a coherent host-microbe signature that discriminated iRBD patients from controls within this discovery cohort (84.8% cross-validated accuracy). These cross-sectional findings characterize candidate fecal signatures associated with iRBD; given the exploratory design and the limited sample size, they are hypothesis-generating, and their value for predicting phenoconversion remains to be established in prospective studies.