Inflammatory bowel disease (IBD) is a multifactorial immune-mediated inflammatory disease of the intestine, comprising Crohn’s disease and ulcerative colitis.
The faecal metabolome offers new avenues for identifying biomarkers for IBD and other immune-mediated inflammatory diseases. Understanding the gut microbiota’s contribution to human metabolism in health and diseases is essential for designing future dietary interventions.
In this study, researchers from the University Medical Centre Groningen, The Netherlands, aimed to determine alterations in the gut metabolism of patients with IBD to pinpoint factors influencing faecal metabolite levels.
Metabolites were assessed in the faecal samples from 238 patients with Chrohn's Disease (CD), 174 patients with Ulcerative Colitis (UC) and 255 non-IBD controls. On average, 1,011 metabolites were detected per sample, ranging from 784 to 1241 molecules.
The findings highlight the potential of faecal metabolites as biomarkers for IBD and show that, despite the influence of lifestyle, genetics and disease, faecal microbes are a strong predictor of the levels and composition of metabolites in the gut.
The report states: "As opposed to colonoscopies, the current invasive gold standard for diagnosing IBD, we demonstrated the potential of faecal metabolites as a non-invasive method for disease diagnosis. The ratio between the levels of two metabolites, lactosyl-N-palmitoyl-sphingosine (d18:1/16:0) and L-urobilin was identified as a biomarker for IBD in our cohort."
In IBD, periods of active disease are characterised by loss of strictly anaerobic bacteria, blooming of facultative anaerobes and alterations in the chemical environment in the gut.
For example, reductions of gut barrier-protecting short-chain fatty acids (SCFA) and alterations in bile acids, sphingolipids and tryptophan-derived metabolites have been consistently reported in faeces of patients with IBD.
However, a large number of molecules in the human body remain uncharacterised, and their implications for human health remain unknown.
Recent technological advances in mass spectrometry techniques have enabled high-throughput characterisation and quantification of a wide range of known and chemically unannotated molecules. In this context, the characterisation of faecal metabolites holds great potential for discovering non-invasive biomarkers and therapeutic targets.
To date, however, studies performing untargeted metabolomics on the faeces of patients with IBD have been scarce, limited in sample size and lacking in-depth information on host genetics, lifestyle, diet and clinical characteristics.
Samples were obtained from two established cohorts: LifeLines, a population biobank from the north of the Netherlands; and 1000IBD, a cohort of patients with IBD from the University Medical Centre of Groningen.
Metabolomics measurements performed by Metabolon (North Carolina, USA) detected 1684 faecal metabolites. The concentrations of eight short-chain and branched-chain fatty acids were measured using liquid chromatography with tandem mass spectrometry.
Linear regression analysis was used to identify microbiome features that differed between controls and IBD. Age, sex, BMI, average bowel movements per day, history of intestinal resections and raw sequencing read depth were included as covariates in the regression models.
Faecal metabolites as novel biomarkers for IBDs
Resulting data revealed alterations in the levels of more than 300 highly prevalent faecal metabolites in patients with IBD. Additionally, the researchers described potential determinants of faecal metabolome composition by integrating untargeted metabolomics with extensive information on dietary habits, host genetics, clinical characteristics and gut microbiota composition.
They note drastic alteration in faecal metabolite composition in patients with IBD suggests a shift from saccharolytic to proteolytic fermentation metabolism, as evidenced by increased levels of metabolites derived from the metabolism of aromatic amino acids, such as p-cresol sulphate (FDRIBD=8.29e–06) and 3-indoxyl sulphate(FDRIBD=0.04). The accumulation of these compounds has been linked to various health conditions, such as chronic kidney disease and colorectal cancer; suggesting that higher presence of these molecules and lower levels of saccharolytic products, like SCFAs, may indicate an unhealthy gut.
The overlap in the faecal metabolite signatures between patients with CD and UC suggests a common underlying alteration in gut metabolism. In total, 58% of the metabolites significantly associated with UC were also found to be associated with CD. When comparing the faecal metabolite profiles of patients with CD and UC, they observed significant differences in the levels of 106 metabolites. For instance, alterations in the bile acid pool were a distinctive feature of CD, while a reduction in the concentrations of SCFAs was a common characteristic of UC.
In patients with CD, researchers observed a marked increase in the faecal levels of sphingolipids, including several sphingomyelins and ceramides. Sphingolipids are components of the intestinal cell membrane and are produced either by the de novo condensation of serine to palmitoyl-CoA or the uptake of endogenous and dietary sphingolipids. In addition to their structural role, sphingolipids can act as signalling molecules, mediating cell differentiation, apoptosis, and inflammation.
In addition to the increased levels of sphingolipids, the researchers also discovered higher levels of N-acylethanolamines (palmitoyl-ethanolamide, linoleoyl-ethanolamide, oleoyl-ethanolamide and stearoyl-ethanolamide) in the faeces of patients with CD compared with non-IBD controls.
Patients with IBD also had elevated levels of long-chain fatty acids (LCFAs) and PUFAs in their faeces. Increasing evidence suggests that diets high in PUFAs can contribute to intestinal inflammation. Exposure to omega-3 and omega-6 PUFA can trigger an inflammatory response in intestinal organoids from patients with CD and in mice models with an impaired glutathione peroxidase 4 (GPX4) gene expression.
The data also revealed a significant increase in the levels of amino acids and their derivatives in patients with IBD. These findings align with previous research conducted on a cohort of newly diagnosed patients with IBD (n=78), where the levels of several amino acids could differentiate IBD samples from controls with high accuracy. In particular, we found a strong increase in taurine levels in IBD samples.
Patients with IBD exhibited depletion of nucleotides, enterolactone (a bacterial product produced from the breakdown of dietary lignans) and biotin (vitamin B7). These findings suggest that the loss of bacterial diversity and biomass in the gut of patients with IBD could drive the reduction in essential functions such as fibre digestion and vitamin production.
The authors suggest the restoration of microbial production of these metabolites through dietary administration of their precursors could serve as a potential strategy to prevent flare-ups and address the dysregulation of the gut microbiome in IBD.
Faecal calprotectin test
Given the substantial variations observed in the metabolite levels between patients with IBD and non-IBD controls, the team investigated the possibility of enhancing the accuracy of the faecal calprotectin test by combining multiple metabolites.
They employed a machine learning approach to predict disease phenotypes.
The predictability of each metabolite was estimated using a combination of host information, dietary habits and the faecal microbiome. Dietary intake predicted the levels of 37 metabolites (>20% of explained variation), with the top 10 dietary-predicted metabolites being 7 unclassified molecules and 3 coffee-related metabolites.
Meanwhile, bacterial abundances were a strong predictor of 82 metabolites (>40% of the variation), including the levels of molecules such as lithocholate (41%, s.d. 18%) and dimethylarginine (ADMA/SDMA, 53%, s.d. 4%). Adding diet and participants’ characteristics slightly improved microbiome-based models.
The researchers acknowledge the limitations of untargeted metabolomics approaches.
The report states: "This study focused on annotated molecules with relatively high prevalence, but a substantial number of metabolites remained unidentified, and their physiological significance is unknown. Approximately one-third of the metabolites detected in our dataset (492 out of 1684 metabolites) could not be linked to a previously characterised compound, emphasising the need for further efforts to fully characterise the molecular diversity in the human body.
"Additionally, the semiquantitative nature of untargeted metabolomics limits the ability to establish the normal concentration range of each metabolite in faeces."
Source: "Faecal metabolome and its determinants in inflammatory bowel disease"
Source: BMJ Gut
"Faecal metabolome and its determinants in inflammatory bowel disease"
Vich Vila A, Hu S, Andreu-Sánchez S, et al