Comparative metabolomics and microbiome analysis of Ethanol vs. OMNImet/gene®•GUT fecal stabilization

Metabolites from feces provide important insights into the functionality of the gut microbiome. As immediate freezing is not always feasible in gut microbiome studies, there is a need for sampling protocols that provide stability of the fecal metabolome and microbiome at room temperature (RT). For this purpose, we investigated the stability of various metabolites and the microbiome (16S ribosomal RNA) in feces collected in 95% ethanol (EtOH) or OMNImet®•GUT/ OMNIgene®•GUT. To simulate in field-collection scenarios, the samples were stored at different temperatures at varying durations (24h +4°C, 24h RT, 36h RT, 48h RT, and 7 days RT), and compared to aliquots immediately frozen at -80°C. We applied several targeted and untargeted metabolomics platforms to measure lipids, polar untargeted metabolites, endocannabinoids, short chain fatty acids (SCFAs), and bile acids (BAs). We found that SCFAs in the non-stabilized samples increased over time, while a stable profile was recorded in sample aliquots stored in 95% EtOH and OMNImet®•GUT. When comparing the metabolite levels between fecal aliquots stored at room temperature and at +4°C, we detected several changes in microbial metabolites, including multiple BAs and SCFAs. Taken together, we found that storing fecal samples at room temperature and stabilizing them in 95% EtOH yielded metabolomic results comparable to flash freezing. We also found that overall composition of the gut microbiome did not vary significantly between different storage types. However, there were notable differences observed in alpha diversity. Taken together, the stability of the metabolome and microbiome in 95 % EtOH provided similar results as the validated commercial collection kits OMNImet®•GUT and OMNIgene®•GUT, respectively. IMPORTANCE The analysis of the gut metabolome and microbiome requires the separate collection of fecal specimens using conventional methods or commercial kits. However, these approaches can potentially introduce sampling errors and biases. In addition, the logistical requirements of studying large human cohorts have driven the need for home collection and transport of human fecal specimens at room temperature. By adopting a unified sampling approach at room temperature, we can enhance sampling convenience and practicality, leading to a more precise and comprehensive understanding of gut microbial function. However, the development and applications of such unified sampling systems still face limitations. The results presented in this study aim to address this knowledge gap by investigating the stability of metabolites and the microbiome (16S ribosomal RNA) from fecal samples collected using 95% EtOH, in comparison to well-established commercial collection kits for fecal metabolome (OMNImet®•GUT) and microbiome (OMNIgene® •GUT) profiling. Additionally, we perform a comparative analysis of various platforms and metabolomic coverage using matrices containing ethanol, evaluating aspects of sensitivity, robustness, and throughput. ### Competing Interest Statement Dr. Kaddurah-Daouk is an inventor on a series of patents on the use of metabolomics for the diagnosis and treatment of CNS diseases and holds equity in Metabolon Inc., Chymia LLC and PsyProtix. All authors declare no competing interests.