Feron, O. | bioRxiv Science

bioRxiv2025

UCL-MetIsoLib: A Public High-Resolution Tandem Mass Spectrometry Library for HILIC-Based Isomer-Resolved Profiling of Glycolysis, Central Carbon Metabolism, and Beyond in Urine, Plasma, Tissues, Cells, and Patient-Derived Organoids

We present UCL-MetIsoLib, a publicly accessible high-resolution tandem mass spectrometry (HRMS/MS) library developed for HILIC-based, ion-pairing free, isomer-resolved metabolomics using a bioinert UHPLC system and the Acquity Premier BEH Amide column. The platform integrates two complementary methods operating under distinct chromatographic conditions (pH 3.5, ESI+; pH 11.0, ESI-), enabling broad metabolic coverage. A total of 334 metabolites are annotated in the library structure, with thiol derivatization incorporated into the extraction protocol to mitigate redox-driven artifacts. Metabolite identification is supported by 245 authentic reference standards and curated according to MSI Level 1 and Level 2 criteria. Validation followed FDA guidelines for bioanalytical method validation and was performed across five biological matrices--urine, plasma, tissues, cultured cells, and patient-derived colorectal organoids--with a U-13C, U-15N-labeled Amino Acid Mixture used as an isotope labeled internal standard. The method demonstrated high precision (<15% RSD intra-/inter-day) and recovery (85-115% across all QC levels). To demonstrate biological applicability, UCL-MetIsoLib was applied to a case study comparing healthy and colorectal cancer-derived organoids. The method enabled confident annotation of metabolite isomers, including key glycolytic intermediates such as DHAP and GA3P, as well as sugar phosphates from the glycolysis and pentose phosphate pathways. Metabolic alterations were observed in tumor organoids, including accumulation of nucleotide derivatives and shifts in central carbon metabolism. These findings emphasize the value of isomer-resolved spectral libraries in detecting biologically meaningful differences that are often missed in conventional untargeted metabolomics workflows.

systems biology↗

bioRxiv2025

Hepatic ADMA/PRMT1 axis regulation is associated with NO-dependent endothelial dysfunction in MASH

Metabolic dysfunction-associated steatohepatitis (MASH) is a severe form of fatty liver disease and a recognized cardiovascular risk factor, yet the mechanisms linking hepatic pathology to vascular dysfunction remain poorly understood. We aimed to investigate whether MASH impairs endothelial function via nitric oxide (NO)-dependent mechanisms and to identify potential liver-derived mediators involved in this process. Endothelial function was assessed in two murine MASH models, Foz mice fed a high-fat diet and C57BL/6JRj mice fed a western diet with fructose, by using wire myography, while blood pressure was monitored via telemetry. NO pathway was further investigated through eNOS expression and activation and Hb-NO measurements. ADMA metabolism was analyzed in both liver tissue and plasma by LC-MS and gene expressions. Additionally, bovine aortic endothelial cells (BAECs) were treated with mouse plasma to measure the circulating factors effects on eNOS activation and the role of oxidative stress. Both models exhibited impaired NO-dependent vasorelaxation without evidence of atherosclerosis. In Foz mice, this impairment was associated with reduced eNOS expression and activation. Surprisingly, plasma Hb-NO levels did not reflect vascular NO deficiency, likely due to elevated hepatic iNOS expression. In both models, hepatic and plasma ADMA levels were increased, concomitant with hepatic upregulation of Prmt1. BAECs exposed to plasma from MASH mice showed reduced eNOS activation independent of oxidative stress. Our findings reveal that MASH is consistently associated with NO-dependent endothelial dysfunction, with ADMA emerging as a key liver-derived mediator. The PRMT1/ADMA/NO axis may represent a mechanistic link between liver pathology and vascular impairment, positioning ADMA as a potential biomarker and therapeutic target for cardiovascular risk associated with MASH. HighlightsO_LIMASH is associated with impaired NO-dependent endothelial function in two distinct MASH models. C_LIO_LICirculating factors disrupt the NOS/NO pathway independently of oxidative stress C_LIO_LIReduced plasma Hb-NO levels do not accurately reflect NO-dependent endothelial dysfunction in a context of MASH. C_LIO_LIElevated plasma and hepatic ADMA levels associated with hepatic prmt1 upregulation are consistently observed in both MASH models. C_LIO_LIThe PRMT1/ADMA/NO axis emerges as a key liver-mediated mechanism driving endothelial dysfunction in MASH C_LI

physiology↗