Periodic Reporting for period 1 - RENAISSANCE (Advanced biomarker discovery for earlier diagnosis and improved prognosis of NAFLD stages via high resolution mass spectrometry.)
Reporting period: 2023-08-01 to 2025-07-31
The RENAISSANCE project addressed a prevalent global health issue: non-alcoholic fatty liver disease (NAFLD), presently being termed as “metabolic dysfunction-associated steatotic liver disease (MASLD)”. MASLD affects approximately 25% of the adult and elderly population worldwide. Early detection is vital for effective intervention before progression to more chronic severe conditions such as metabolic dysfunction-associated steatohepatitis (MASH), cirrhosis, or hepatocellular carcinoma (HCC). The current clinical diagnosis is based on imaging techniques such as ultrasonography and elastography which, however, are not available to all in need, while robust and valid laboratory markers of MASLD are lacking for more characteristic diagnosis. Therefore, RENAISSANCE aimed to evaluate small-molecule metabolites and proteins as the potential markers for prognosis of MASLD stages. The project applied state-of-the-art mass spectrometry instrumentation for comprehensive profiling of small molecule metabolites and proteins related to nutrition and inflammation, which play critical role in the pathogenesis and severity of MASLD. The project has made significant progress towards its scientific impact, and generated results culminating from omics studies will contribute to understanding the underlying pathological molecular processes and metabolism in MASLD progression.
The project adopted a holistic approach of metabolomics and proteomics profiling using mass spectrometry. The study addressed the following primary research objectives: (i) targeted assessment of tryptophan pathways metabolites, (ii) targeted assessment of inflammatory proteins, (iii) non-targeted serum metabolomics, and (iv) non-targeted metabolomics analysis of serum-derived exosomes. Individual assays were applied to achieve these research objectives.
Blood-based assays were developed and optimized under the designed objectives, with pooled serum and plasma samples used for method development and validation. A rapid targeted method with a high coverage has been developed for the quantification of tryptophan pathway metabolites in serum and plasma using UHPLC-MS/MS. Tryptophan pathway metabolites play a key role in gut-microbiota mediated metabolic mechanisms and are indicative of diet and co-microbial functions, therefore the assay has high significance to MASLD and intervention, with regards to the nutritional influence on risk and treatment outcomes. Within the proteomic profiling, 33 inflammatory, immunological, and lipid-related proteins were quantified using a targeted UHPLC/SRM-MS quantitative assay. These quantitative measurements of metabolites and proteins under targeted assays are significant to determine the concentrations in the biosamples. The developed method has been applied for corresponding evaluations of available MASLD cohort samples, and further data analysis is in progress for determining conclusive outcome. However, differential expression of these metabolites and proteins could be significant to represent promising candidates for molecular biomarkers of the disease progression and understanding their role in biological processes like metabolism.
Non-targeted serum metabolomics was performed using GC-Orbitrap-HRMS, while non-targeted metabolomics of serum-derived exosomes involved application of a pilot workflow for exosome isolation, molecular characterization, and metabolomics profiling on a UHPLC-Orbitrap-HRMS instrument. These studies lead to identification and characterization of perturbed endogenous metabolites and associated metabolic pathways in analysed serum samples and help to distinguish variable features pertaining to distinct MASLD stages. Furthermore, the findings will expand current knowledge by identifying additional metabolites not previously reported as perturbed in this disease.
Blood-based assays were developed and optimized under the designed objectives, with pooled serum and plasma samples used for method development and validation. A rapid targeted method with a high coverage has been developed for the quantification of tryptophan pathway metabolites in serum and plasma using UHPLC-MS/MS. Tryptophan pathway metabolites play a key role in gut-microbiota mediated metabolic mechanisms and are indicative of diet and co-microbial functions, therefore the assay has high significance to MASLD and intervention, with regards to the nutritional influence on risk and treatment outcomes. Within the proteomic profiling, 33 inflammatory, immunological, and lipid-related proteins were quantified using a targeted UHPLC/SRM-MS quantitative assay. These quantitative measurements of metabolites and proteins under targeted assays are significant to determine the concentrations in the biosamples. The developed method has been applied for corresponding evaluations of available MASLD cohort samples, and further data analysis is in progress for determining conclusive outcome. However, differential expression of these metabolites and proteins could be significant to represent promising candidates for molecular biomarkers of the disease progression and understanding their role in biological processes like metabolism.
Non-targeted serum metabolomics was performed using GC-Orbitrap-HRMS, while non-targeted metabolomics of serum-derived exosomes involved application of a pilot workflow for exosome isolation, molecular characterization, and metabolomics profiling on a UHPLC-Orbitrap-HRMS instrument. These studies lead to identification and characterization of perturbed endogenous metabolites and associated metabolic pathways in analysed serum samples and help to distinguish variable features pertaining to distinct MASLD stages. Furthermore, the findings will expand current knowledge by identifying additional metabolites not previously reported as perturbed in this disease.
The overarching goal of the project was to follow multi-omics approach for assessing small metabolites and proteins to represent promising candidates for potential biomarkers of the disease diagnosis and progression through blood-based analysis. The results will contribute to better healthcare solutions and more efficient use of resources in disease management. The advancement in new methodologies and results related to omics studies will be useful to support analytical resources and application in relevant studies across omics research field. In the long term, more accurate characterization of MASLD stages could reduce the clinical burden supporting better treatment and disease management, thus the outcome will be meaningful at the societal level as well.