To Örebro University

Intestinal luminal microbial metabolites affect tryptophan and serotonin metabolism, and cross or modify the blood-brain barrier (BBB). Understanding those mechanisms further necessitates integrated gut-brain axis model systems. Using an ex vivo-in vitro approach, H2O2-stressed or non-stressed human dermal fibroblasts - representing the BBB - are cultured with serosal fluids of healthy or irritable bowel syndrome human colonic biopsies collected from Ussing chamber experiments, after participant's colon was exposed to butyrate in vivo, fecal fiber fermentation or control supernatant ex vivo. Culturing fibroblasts with serosal fluids does not compromise viability or have cytotoxic effects. Serosal fluids alone do not alter expression of tryptophan-related large amino acid membrane transporter genes and proteins, nor their activity (i.e., tryptophan uptake). However, adding serosal fluids to fibroblasts prior to oxidative stress indicate a protective role. This new model allows investigation of direct effects of serosal content on BBB-representing fibroblasts and is highly promising for more personalized applications.

Shifting to a plant-based diet naturally alters protein source choices. In many countries, protein from yellow pea is widely used as a main ingredient in meat alternatives. Still, its biological effects, especially regarding gastrointestinal health, remain incompletely understood. The aim of our study was to investigate how a weekly increase in the intake of a well-characterized pea protein isolate affects surrogate markers of health, fecal short-chain fatty acids and gut microbiota composition in healthy individuals. Male and female adults (N = 29) participated in this exploratory intervention study. A 4-week pre-intervention period for questionnaires and fecal samples collection was followed by a 4-week supplementation. Participants consumed isolated pea protein in weekly increasing amounts, starting from 0.25 g per kg body mass per day in week 5 to 1.00 g per kg body mass per day in week 8. Questionnaire data, fecal samples as well as fasting blood and 24 h urine samples were collected weekly. Data from biological samples and questionnaires confirmed a healthy study population and compliance. Fecal calprotectin levels significantly increased only in a subset of participants, which was accompanied by higher fecal water cytotoxicity in vitro. Short-chain fatty acids mainly rose in those subjects with stable calprotectin levels. Relative abundances of Limosilactobacillus frumenti, Odoribacter splanchnicus and Lactobacillus crispatus increased significantly in the total population during the intervention while the relative abundance of Bifidobacterium longum and Bifidobacterium catenulatum decreased. Our results indicate that an increased intake of pea protein isolate affects the growth of certain beneficial bacterial strains and differentially influences markers related to gut inflammation in healthy individuals.

Perfluorohexyloctane (F6H8) is a semifluorinated alkane recently approved for ophthalmic treatment of dry eye disease. Although considered locally safe for topical use, its structural similarity to persistent per- and polyfluoroalkyl substances (PFAS) raises concerns about systemic accumulation and long-term toxicity. To investigate potential hepatic effects, we examined the metabolic impact of F6H8 exposure in human HepaRG hepatocytes across a broad concentration range representing short- and long-term exposure scenarios. Combined targeted and untargeted metabolic profiling by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOFMS) was performed on intracellular extracts and extracellular media. F6H8 induced pronounced, concentration-dependent metabolic alterations, many of which exhibited non-monotonic responses. Low concentrations primarily affected amino acid, fatty acid, and lipid metabolism, while central carbon metabolism was disrupted only at the highest exposures. Notably, a putative biotransformation product, perfluorohexyloctanoic acid, was detected, suggesting metabolic persistence and conversion to a PFAS-like structure. This metabolite showed strong associations with cellular metabolic profiles and elicited metabolic changes that only partially overlapped with those induced by the parent compound, indicating distinct biological activity following biotransformation. These findings indicate that F6H8 elicits broad metabolic reprogramming and may not be metabolically inert as previously assumed. Given its clinical use and structural similarity to persistent fluorochemicals, the results highlight the need for comprehensive, long-term safety assessment of F6H8 and related semifluorinated alkanes.

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental pollutants with known bioaccumulative properties and increasing recognition for their potential to disrupt metabolic pathways, including bile acid transport and lipid metabolism. Despite growing global attention, data on PFAS exposure and distribution in the Balkan region, including Serbia, remain limited. This work presents preliminary findings on PFAS presence and explores potential associations with bile acid metabolism in patients with metabolic dysfunction-associated steatotic liver disease (MASLD) and age-, BMI-, and sex-matched controls, as part of a broader multi-omics investigation. Plasma samples were collected from 122 participants (MASLD patients and matched controls) and metabolites were extracted using a monophasic solution of methanol, methyl tert-butyl ether, and isopropanol (20:15:15) containing internal standards. Extracts were analyzed using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry on a C18 column. Data processing was performed using MZmine 3 software, and compound annotation was based on an in-house spectral library. Among the detected environmental contaminants, three PFAS compounds—perfluorohexane sulfonic acid (PFHxS) and both linear and branched isomers of perfluorooctane sulfonic acid (PFOS)—were consistently identified across all samples, suggesting widespread low-level exposure in the cohort, with no significant differences in PFAS abundance between MASLD patients and healthy controls. These preliminary results highlight the presence of PFAS compounds in all participants, reflecting a baseline environmental exposure within this population. Although no clear associations with MASLD status were observed, the integration of environmental contaminant data into multi-omics frameworks is critical for uncovering subtle metabolic perturbations. Ongoing analyses will focus on profiling secondary bile acids and microbiota-derived metabolites to explore potential downstream effects of PFAS exposure and their relevance to MASLD pathophysiology.

Dietary fibers (DF) from plant-based foods promote health benefits through their physicochemical properties and fermentation by the gut microbiota, often studied in relation to changes in gut microbiota profile and production of gut microbiota-derived metabolites. Here, we characterized structural motifs (i.e., oligomers) produced during DF breakdown upon colonic fermentation and explored their interaction with toll-like receptors (TLRs) present on the surface of human intestinal and immune system cells. Wheat arabinoxylan (WAX) was subjected to in vitro colonic fermentation, with its structural motifs identified and tracked throughout the fermentation process. Using carbohydrate-active enzymes, six well-defined fractions of arabinoxylans and linear xylans identified during colonic fermentation were produced and tested for interaction with tool-like receptors (TLR)2 and TLR4 via reporter cell assay. The results showed structure-dependent effects, with TLR2 inhibition and TLR4 activation varying based on the degree of polymerization and branching. Molecular docking confirmed that minor structural changes in oligomers structure significantly influenced these interactions. The study supports the hypothesis that oligomers and polysaccharides affect cell receptors through complex, multi-receptor interactions, and highlights the potential for enzymatic tailoring of DF to create functional ingredients with targeted effects on human health.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most prevalent chronic liver disease in the Western world, and is rapidly increasing worldwide. Serbia, along with other Balkan countries, faces particularly high rates of MASLD, yet remaining underrepresented in large-scale European research initiatives, including the MASLD registry. The absence of structured clinical and molecular datasets from this region emphasize the need for locally conducted, population-specific studies. This work presents preliminary results from the first comprehensive multi-omics study of MASLD in the population from Serbia, aiming to identify a lipidomic signature of liver dysfunction and to explore microbial metabolites potentially involved in disease pathophysiology, using both untargeted and targeted metabolomics approaches. Plasma and fecal samples were collected from 122 participants, including MASLD patients and sex-, age-, and BMI-matched healthy controls. Lipids from plasma, as well as semi-polar and polar metabolites, were extracted using a monophasic extraction solution (MeOH:MTBE:IPA (20:15:15)) containing internal standards. The samples were analyzed using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS, Agilent Technologies, Santa Clara, CA, USA). The analysis was performed on an ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, particle size 1.7 µm; Waters, Milford, USA). Data were processed and analyzed using MZmine 3 software, with lipid and metabolite identification based on an in-house library. A total of 1751 lipids and 1315 semi-polar/polar metabolites were detected in plasma samples, with 214 lipids and 132 semi-polar/polar metabolites identified. Principal component analysis did not reveal significant separation between MASLD patients and healthy controls. At the lipid class level, however, distinct clustering patterns emerged, pinpointing alterations in lipid metabolism. Ongoing analyses aim to further refine these observations and investigate associations with clinical parameters, with the goal of uncovering meaningful patterns that could contribute to the discovery of disease biomarkers and the elucidation of pathophysiological mechanisms.

Mass spectrometry-based methods have become fundamental to exposome research, providing the capability to explore a broad spectrum of chemical exposures. Liquid and gas chromatography coupled with low/high-resolution mass spectrometry (MS) are among the most frequently employed platforms due to their sensitivity and accuracy. However, these approaches present challenges, such as the inherent complexity of MS data and the expertise of biologists, chemists, clinicians, and data analysts to integrate and interpret MS data with other datasets effectively. The "omics" era advances rapidly, driven by developments of AI-based algorithms and an increase in accessible data; nevertheless, further efforts are necessary to ensure that exposomics outputs are comparable and reproducible, thus enhancing research findings. This review outlines the principles of MS-based methods for the exposome analytical pipeline, from sample collection to data analysis. We summarize and review both standard and cutting-edge strategies in exposome research, covering sample preparation, focusing on MS-based platforms, data acquisition strategies, and data annotation. The ultimate goal of this review is to highlight applications that enable the simultaneous analysis of endogenous metabolites and xenobiotics, which can help enhance our understanding of the impact of human exposure on health and disease and support personalized healthcare.

Advancing our understanding of human health and disease requires comprehensive analytical approaches capable of capturing the complex interplay between endogenous metabolism and environmental exposures. A major challenge in clinical research is the ability to capture multidimensional data, particularly a broad range of biochemical profiles, due to limitations of biological resources, time, and budget. In this study, we introduce the SIMPLIFY Protocol, a unified monophasic extraction method that enables the simultaneous extraction of chemical exogenous products and endogenous molecules. The method was evaluated against in-house extraction techniques, including protein precipitation with methanol (MeOH) and acetonitrile (ACN), and the Folch method using various sample types, particularly certified reference materials. We demonstrate that the SIMPLIFY Protocol not only performs comparably to our in-house methods but also offers enhanced versatility for additional applications such as derivatization and proteomics. The analyte abundances and reproducibility with this method strongly correlate with those from in-house-established techniques across diverse sample types. The method encompasses a broad spectrum of compounds, effectively profiling approximately 800 identified compounds, including polar compounds (e.g., amino acids), semipolar compounds (e.g., polyfluorinated compounds, bile acids, and lysophophatidylcholine), and nonpolar compounds (e.g., cholesteryl ester), with some limitations in extracting triacylglycerols. By maintaining simplified workflow and minimizing biological and resource consumption of multiple extractions, this method supports high-throughput exposomics/metabolomics and lipidomics studies. Furthermore, its streamlined design facilitates (semi)automation, making it highly suitable for large-scale clinical studies, where efficiency, cost-effectiveness, and sample availability are critical factors.

Oligosaccharides represent an important class of bioactive compounds with diverse structure-dependent functionalities. While traditionally investigated for prebiotic effects on gut microbiota, increasing evidence shows that they can also interact directly with intestinal and immune cell receptors (e.g., pattern recognition receptors, C-type lectin receptors, G-protein coupled receptors, and scavenger receptors), influencing inflammation, mucosal immunity, and epithelial barrier function. In a circular bioeconomy context, oligosaccharides offer an underexplored sustainable route for upcycling agro-industrial and forestry biomass by converting polysaccharides into structurally tailored products. This review synthesizes current knowledge on the direct effects of plant-derived oligosaccharides on host receptors and describes enzymatic and green chemistry-based methods for their production and extraction. We propose a new roadmap for oligosaccharide upcycling using assessments for their biological effects, characterization, standardization, and alignment with regulatory aspects. This enables translating receptor-active oligosaccharides from biomass utilization into innovative solutions for precision nutrition and sustainable functional food development.

Dietary fibre intake is essential for all human beings and has been correlated to beneficial health effects. Pea hull fibres (PF) are generally seen as a side stream during extraction of protein and starch from yellow pea but could be used in various food products to boost fibre content. In this study, the thermal treatment of pea hull fibres was investigated in terms of physicochemical properties and in vitro colonic fermentation. The PF that was subjected to heating showed an increase of fibres solubilised in the liquid and particle size. Results also showed that viscosity and storage modulus increased with thermal treatment, possibly due to the swelling of the PF. The pea fibre was readily fermentable based on total gas production and pH. However, the susceptibility to fermentation of PF did not increase with thermal treatment. Total gas production and short chain fatty acid produced were similar independent of thermal treatment. Conclusively, heating of the PF resulted in increased ability to structure water suspension, owing to increased fibre particle size, but is not sufficient to increase short chain fatty acid production during colonic fermentation. To explain this, we propose that the changes in cell wall structure were not major enough to induce higher fermentability.