Gut microbial metabolites, short-chain fatty acids (SCFAs), are found at multiple locations in the host body and are identified as important metabolites in gut microbiome-associated diseases. Quantifying SCFAs in diverse biological samples is important to understand their roles in host health. This study developed an accurate SCFA quantification method by performing gas chromatography–mass spectrometry (GC/MS) in human plasma, serum, feces, and mouse cecum tissue. The samples were acidified with hydrochloric acid, and the SCFAs were extracted using methyl tert-butyl ether. In this method, distilled water was selected as a surrogate matrix for the quantification of SCFAs in target biological samples. The method was validated in terms of linearity, parallelism, precision, recovery, and matrix effect. The developed method was further applied in target biological samples. In conclusion, this optimized method can be used as a simultaneous SCFA quantification method in diverse biological samples.

Circulatory lipids are important markers for characterizing disease phenotypes; however, accurately determining lipid species remains a significant challenge in lipidomic analysis. Here, we present a novel analytical workflow for accurate lipidome characterization in human plasma using mass spectrometry (MS) through the integration of hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC). This workflow enables rapid screening of 1,966 lipid species across 18 lipid classes using HILIC-multiple reaction monitoring (MRM), which enables facile identification of lipid species by lipid class-based separations. In the NIST Standard Reference Material for Human Plasma (SRM 1950), 489 lipid species were identified using HILIC-MRM and subsequently analyzed with RPLC-parallel reaction monitoring (PRM) to resolve potential lipid isobars within the same lipid class. Notably, RPLC-PRM identified 70 additional lipidomic features in SRM 1950 that were not detectable with HILIC-MRM. Furthermore, a high correlation (Pearson correlation coefficient = 0.81) was observed regarding the concentrations of lipid species not carrying isobaric interferences in between HILIC-MRM and RPLC-PRM, indicating that the individual lipid concentrations measured by each platform can be integrated. The workflow was further applied to a cohort of 284 human plasma samples from chronic kidney disease (CKD) patients, successfully profiling lipidomic phenotypes across CKD subtypes. These findings demonstrate that combining HILIC-MRM and RPLC-PRM as complementary platforms enhances the accuracy and comprehensiveness of lipidomic analysis.

Studies on the immune-regulatory roles played by the commensal microbes residing in the nasal mucosa consider the contribution of antiviral immune responses. Here, we sought to identify the nasal microbiome, Staphylococcus epidermidis-regulated antiviral immune responses and the alteration of polyamine metabolites in nasal epithelium. We found that polyamines were required for the life cycle of influenza A virus (IAV) and depletion of polyamines disturbed IAV replication in normal human nasal epithelial (NHNE) cells. Inoculation of S. epidermidis also suppressed IAV infection and the concentration of polyamines including putrescine, spermidine, and spermine was completely attenuated in S. epidermidis-inoculated NHNE cells. S. epidermidis activated the enzyme involved in the production of ornithine from arginine and downregulated the activity of the enzyme involved in the production of putrescine from ornithine in nasal epithelium. S. epidermidis also induced the activation of enzymes that promote the extracellular export of spermine and spermidine in NHNE cells. Our findings demonstrate that S. epidermidis is shown to be able of creating an intracellular environment lacking polyamines in the nasal epithelium and promote the balance of cellular polyamines in favor of the host to restrict influenza virus replication.

Kim H, et al. Transl Clin Pharmacol. 2025 Jun;33(2):66-79. https://doi.org/10.12793/tcp.2025.33.e7

The commensal nasal microbiota Staphylococcus epidermidis (S. epidermidis) can suppress influenza A virus (IAV) activity in the host lung by modulating the microcellular environment. However, the metabolic mechanisms underlying this antiviral effect remain unclear. This study employs a novel combination of atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) and Orbitrap secondary ion mass spectrometry (OrbiSIMS) imaging to investigate the metabolic interactions between nasal microbiota and IAV infection in mouse lung tissue. By utilizing this high-resolution dual-platform MS imaging (MSI) approach, we achieved high spatial resolution mapping of metabolic alterations, revealing polyamine metabolism as a key pathway mediating the antiviral effects of S. epidermidis. Our findings demonstrate that S. epidermidis inoculation downregulates polyamine metabolism, significantly suppressing IAV replication, a mechanism confirmed through targeted polyamine supplementation. Additionally, sulfate/sulfite metabolism was identified as a crucial metabolic pathway modulated in IAV-infected and S. epidermidis-treated lungs. This study represents the first application of integrated AP-MALDI and OrbiSIMS MSI to antiviral metabolomic analysis, providing critical insights into host-microbiota interactions in respiratory infections. Our results provide a foundation for utilizing spatial metabolomics to explore novel therapeutic targets for viral infections and highlight the potential of nasal microbiota in respiratory defense. These findings could advance MS-based biomarker discovery and antiviral drug development.