Arq Bras Cir Dig abcd ABCD. Arquivos Brasileiros de Cirurgia Digestiva (São Paulo) ABCD, arq. bras. cir. dig. 0102-6720 2317-6326 Colégio Brasileiro de Cirurgia Digestiva RESUMO RACIONAL: A doença inflamatória intestinal (DII), que engloba a doença de Crohn (DC) e a colite ulcerativa (CU), carece de etiologia conhecida. Embora sintomas clínicos, imagens e colonoscopia sejam ferramentas diagnósticas comuns, a calprotectina fecal (CalF) atua como biomarcador amplamente utilizado para monitorar a atividade da doença. A metabolômica, no âmbito das ciências “omics,” apresenta promissoras perspectivas para identificação de biomarcadores de progressão da doença. Este método envolve o estudo de metabólitos em meios biológicos para revelar fatores patológicos. OBJETIVOS: Explorar a metabolômica fecal em pacientes com DII, avaliando seu potencial na diferenciação de subtipos e na avaliação da atividade da doença usando CalF. MÉTODOS: Estudo transversal incluindo pacientes com DII, dados clínicos e medições de CalF (=200μg/g como indicador de doença ativa). RESULTADOS: A metabolômica fecal utilizou cromatografia de massa/extração em fase sólida com o software MetaboAnalyst 5.0 para análise. Dos 52 pacientes (29 CU, 23 DC), 36 (69,2%) apresentaram atividade inflamatória. Foram identificados 56 metabólitos fecais, sendo os ácidos hexadecanoico, esqualeno e octadecanoico notáveis na distinção entre CD e CU. Para CU, os ácidos octadecanoico e hexadecanoico correlacionaram-se com a atividade da doença, enquanto o ácido octadecanoico foi mais relevante em CD. CONCLUSÕES: Os achados destacam o potencial da metabolômica como um complemento não invasivo para avaliar a doença inflamatória intestinal, auxiliando no diagnóstico e na avaliação da atividade da doença. INTRODUCTION Crohn’s disease (CD) and ulcerative colitis (UC), entitled inflammatory bowel diseases (IBD), are chronic diseases that cause inflammation in the gastrointestinal tract and extraintestinal complications, which share similar symptoms like abdominal pain, diarrhea, and weight loss, but differ in their extent, location, complications, and prevalence26,28. Because of its impact on patients’ quality of life and work activities, IBD represents a significant public health problem. Treatment aims to achieve remission through a multifaceted approach that includes both nonpharmacological interventions like psychological and nutritional support, and pharmacological interventions like immunosuppressants and anti-inflammatories, as well as traditional surgical interventions32,36. The costs for the health system are considered high and are due to multiple factors such as diagnostic, medications, hospitalization, surgeries, and absenteeism from work8,16. The etiology of IBD is unknown, but the interaction between genetics, immunological, and environmental factors, such as microbiota, contribute to its development16. In this context, intestinal dysbiosis - the imbalance of the intestinal microbiota - is closely related to the pathogenesis and clinical complications of IBD; however, it is still not possible to distinguish whether this dysbiosis is a cause or a consequence of the disease2,38. Thus, microbiota and dysbiosis are currently the subject of important investigations in the field of diagnosis and therapy in IBD. The diagnosis of IBD typically involves invasive and expensive measures such as colonoscopy and histopathological evaluation, in addition to clinical assessment, which can be uncomfortable for patients. In recent years, studies have aimed to identify laboratory markers with high sensitivity and specificity for noninvasive diagnosis and monitoring of these diseases7. Fecal calprotectin (FC), a calciumand zinc-binding protein found mainly within neutrophils, has been presented as an effective, low-cost, and highly reproducible marker for both diagnosis and follow-up of IBD7. FC correlates well with the top endoscopic assessment scores used in clinical practice [Simple Endoscopic Score for Crohn’s Disease (SES-CD), Ulcerative Colitis Score (UCIS), and Mayo Score)34. However, its low sensitivity (83%) and specificity (60%) make it a nonspecific inflammatory biomarker. Therefore, definitive diagnosis still requires biopsies collected through colonoscopy, which carries a risk of pain, bleeding, and perforation7. Metabolomics, an omic technique, has been recently studied in several diseases, including IBD. It has been considered a high sensitivity and specificity method for assessing endoscopic activity using an FC cutoff of 200 μg/g of stool. Additionally, several metabolites identified by metabolomic techniques, such as tricarboxylic acid cycle (TCA) cycle intermediates, amino acids, fatty acids, glycerophospholipids, glycerolipids, prenol lipids, sterol lipids, choline, and sphingolipids in serum, urine, feces, or intestinal biopsies, are typically involved in molecular pathways implicated in the inflammatory response. This reinforces the diagnostic potential of metabolomic analysis3. Despite increasing research on omic sciences in IBD, several questions remain unanswered, such as their use in the differential diagnosis of CD and UC, as well as their use in assessing the therapeutic potential of pharmacological and nonpharmacological treatments. Therefore, this study aims to identify metabolites present in the feces of individuals with IBD and correlate them with disease activity, as measured by levels of FC. METHODS Study design, participant selection, inclusion criteria, and ethical oversight This study employs a cross-sectional design, with patient recruitment carried out via convenience sampling. The participant pool includes individuals of all genders and age ranges, diagnosed with IBD, who are under the care of the coloproctology outpatient clinic at University Hospital Professor Alberto Antunes, Universidade Federal de Alagoas, Brazil. Patients who accepted the invitation to participate in the study were provided with clear instructions regarding the proper collection of fecal samples. These samples were subsequently submitted during their subsequent clinic visit for the evaluation of both FC levels and fecal metabolites. Conducting the study in strict accordance with the ethical principles set forth in the Declaration of Helsinki, it received formal endorsement from the Ethics and Research Committee of the Federal University of Alagoas (Approval Number: 2725386). This rigorous ethical oversight underscores the commitment to safeguarding the welfare and rights of all involved participants. Fecal calprotectin analysis FC was analyzed using the BÜHLMANN Quantum Blue® device. Stool samples were placed in extraction tubes and diluted with a 1:16 ratio of BÜHLMANN fCAL® kit solution to extraction buffer. The resulting mixture was vortexed for 1 min and then centrifuged at 3000×g for 5 min. The final solution was subsequently analyzed using the instrument for a duration of 15 min. FC levels were classified as altered (high) when they reached=200 μg/g of feces5. Analysis of volatile organic compounds Volatile organic compounds (VOCs) present in donors’ fecal samples were analyzed using gas chromatography mass spectrometry/solid phase microextraction (GC-MS/SPME) technique, as previously described1. Statistical analysis The statistical analysis was performed using the SPSS® version 26 software. Continuous variables were expressed as mean±standard deviation (SD) and categoric variables as frequency [n (%)]. Frequency comparison using the chi-square test/Fisher tests, according to CD/UC, and t-test was used to compare means. Significance was considered when the p<0.05. The online platform MetaboAnalyst 5.0 (available free of charge at https://www.metaboanalyst.ca) was utilized to conduct all statistical analyses requiring computational support. This software, known for its widespread use and recommendation in this field, contributes to numerical precision and scientific reproducibility. Chromatogram data obtained through GC were transformed into a data matrix using Chromeleon 7.2 software (equipment program). Regions containing inferential signals were excluded from the analysis. The concentrations of identified metabolites were normalized by the median, followed by logarithmic transformation (base 10) and scaling using the Pareto method. Supervised partial least squares discriminant analysis (PLS-DA) was conducted to discern metabolite variations among stool sample groups. PLS-DA, a supervised technique utilizing multivariate regression information, predicts class associations. Variations in sample metabolites were calculated using the variable importance on projection (VIP) measure. This score represents a weighted sum of squares of PLS loadings, accounting for the explained Y-variance of each component. In this study, VIP measurements above 2.0 were considered significant. RESULTS Fifty-two patients with IBD, 29 with UC and 23 with DC, were studied. Of these patients, 38 were women and 14 were men, with a mean age of 41 years (UC=43.0±15.7, and CD=39.7±16.2; p=0.463, p>0.05), the youngest being 12 years old and the oldest being 69 years old. Thirty-six patients (69.2%) had high FC and had no statistical difference between the prevalence of inflammation detected by FC levels and the type of IBD (p>0.05). There was no significant difference in the overall demographic and clinical characteristics of the study participants concerning the type of IBD, as indicated in Table 1. Table 1 Demographic and clinical characteristics according to inflammatory bowel disease source. Totaln (%) Ulcerative colitisn (%) Chron diseasen (%) p-value Sex Female 38 (73.1) 22 (75.9) 16 (69.6) 0.611 Male 14 (26.9) 7 (24.1) 7 (30.4) Race Not white 47 (90.4) 27 (93.1) 20 (87.0) 0.644 White 5 (9.6) 2 (6.9) 3 (13.0) Smoking Yes 5 (9.6) 4 (13.8) 1 (4.3) 0.368 No 47 (90.4) 25 (86.2) 22 (95.7) Alcohol consumption Yes 5 (9.6) 1 (3.4) 4 (17.4) 0.157 No 47 (90.4) 28 (96.6) 19 (82.6) Comorbidities Yes 24 (46.2) 12 (41.4) 12 (52.2) 0.438 No 28 (53.8) 17 (58.6) 11 (47.8) Surgery Yes 25 (48.1) 15 (51.7) 10 (43.5) 0.544 No 27 (51.9) 14 (48.3) 13 (56.5) Extraintestinal manifestations Yes 35 (67.3) 18 (62.1) 17 (73.9) 0.366 No 17 (32.7) 11 (37.9) 6 (26.1) Values are presented as numbers (%). Seventy-four metabolites were found through the mass spectrometry library (Chromeleon 7.2). Metabolites were compared with data in the Human Metabolome Database (available at https://hmdb.ca), 18 of which were unidentified (Unknown). The 56 identified metabolites were: p-cresol, 2-undecanone, undecanal, indoleacetic acid, ethyl decanoate, ethyl-heptane, squalene, 1-decene, naphthalene, pentadecane, trimethyl-dodecathyene, tetradecanal, dodecanoic acid, dodecanoic-ethyl acid -ester, methyl amine, hexadecane, cyclooctasiloxane, dodecanol, trimethyl amine, pentadeicin, pentadecanone, nonadecene, tridecan-1-ol, tricosene, tetradecanoic acid, undecanone, pentadecanoic acid, hexadecanol, heptadecanone, squalene, pentadecanal, cyclodecasiloxone, docosene, hexadecanoic acid (palmitic acid), hexanoic acid, 9-octadecenal, cis-11-hexadecenal, octadecenal, 9-Octadecen-1-ol, linoleoyl chloride, octadecanol, oleic acid, cis-9-hexadecenal, octadecanoic acid (stearic acid), 11-hexadecen-1-ol, cyclononasiloxane, 1-hexadecanol, cyclopentadecane, heptacosane, cyclodecasiloxane, octadecane, docosane, 11-butyl-, quantacure ITX, heptacosane, and octacosane. Figure 1 shows the result of the partial PLS-DA of samples between DC and UC. The points in Figure 1 represent the metabolic profile of each patient. Thus, patterns of separation can be seen in the data sets. There are patients with distinct profiles between UC and CD, and some in intersection areas where there are metabolites with variations in common between the two groups. The method has a good accuracy (0.60) in groups of up to five primary metabolites. Figure 1 Patterns of separation between Crohn’s disease (CD) and ulcerative colitis (UC) by partial least squares discriminant analysis (PLS-DA). The green area corresponds to patients with CD, and the pink area corresponds to patients with UC. Variables in projection (VIP) score (B). Figure 1 (A) Patterns of separation between Crohn’s disease and ulcerative colitis by partial least squares discriminant analysis. The green area corresponds to patients with Crohn’s disease, and the pink area corresponds to patients with ulcerative colitis. Variables in projection score (B). UC: ulcerative colitis; CD: Crohn’s disease; VIP: variables in projection. The VIP score classification of the components of the CD and UC groups is shown in Figure 1. It includes the most significant variations of metabolites that discriminate the UC and CD groups by the PLS-DA. Considering necessary VIP measurements above 2.0 as pre-established in the methodology of the work, the metabolites found were (highest score in the group UC): hexadecanoic acid, squalene, and octadecanoic acid. These three mentioned metabolites were used to discriminate the groups through the receiver operating characteristic (ROC) curve. The area under the curve (ACU) of the model was 0.765 (95% confidence interval [CI]: 0.35-1.0). The relationship between the FC and the UC groups can be observed in Figure 2. The separation standards for the datasets showed good accuracy (0.65). The main metabolites found in this analysis were as follows: octadecanoic acid and hexadecanoic acid had the highest score in the group >200 μg/g, whereas 11-hexadecen-1 and pentadecanone had the lowest score in the group <200 μg/g. The ROC curve using such metabolites has resulted in an ACU of 0.77 within the confidence interval (95%CI 0.37-0.98). Figure 2 (A) Ulcerative colitis: patterns of separation between fecal calprotectin (<200 μc/g of feces vs. =200 μc/g of feces) by partial least squares discriminant analysis. The green area corresponds to patients with Crohn’s disease, and the pink area corresponds to patients with ulcerative colitis. Variables in projection score (B). VIP: variables in projection. Figure 3 shows metabolites that differentiate disease activity in CD and the separation standards for the datasets showed good accuracy (0.60). The main metabolites found in this analysis were: octadecanoic acid and cyclopentadecanoic acid demonstrated the highest score in the group >200 μg/g, whereas cyclodecasiloxone and oleic acid-1 had the highest score in the group <200 μg/g. The ROC curve, using such metabolites, shows an ACU of 0.85 within the confidence interval (95%CI 0-1). Octadecanoic acid, as in the CD group, is the most relevant metabolite in patients with disease activity. Figure 3 (A) Crohn’s disease: patterns of separation between fecal calprotectin (<200 μc/g of feces vs. =200 μc/g of feces) by partial least squares discriminant analysis. The green area corresponds to patients with Crohn’s disease, and the pink area corresponds to patients with ulcerative colitis. Variables in projection score (B). VIP: variables in projection. DISCUSSION This study has provided significant insights into the distinction of intestinal metabolites found in the feces of patients with CD or UC. Additionally, it corroborated that this composition is directly influenced by the degree of disease activity, as assessed through FC levels, a noninvasive biomarker widely employed in clinical practice. Metabonomics serves as a quantitative approach for analyzing metabolites and establishing associations between metabolites and physiological as well as pathological changes40, rendering it a potent tool in the exploration of human health9. Metabolite analyses are commonly conducted utilizing noninvasive sample sources, such as fecal samples24, urine41, blood11,20, and tissue extracts4 not requiring invasive procedures. In this investigation, fecal samples were subjected to analysis using mass spectrometry coupled with the GC-MS technique, leading to the identification of a distinct IBD metabolome signature. Discernible variations in metabolite patterns were observed between CD and UC patients. Specifically, the latter exhibited elevated levels of saturated long-chain fatty acids (SLCFAs) such as hexadecanoic acid, octadecanoic acid, triterpenoids, and squalene. The differentiation of fecal metabolomics between individuals with IBD and healthy controls17,18,23,25,35,37 appears to be more conspicuous than the distinction between CD and UC subtypes. Studies conducted by Marchesi et al.25, Garner et al.13, and Bjerrum et al.6 successfully identified differences between CD and UC, akin to the findings in our study6,13,25. However, Santoru et al.35 and Franzosa et al.12 were unable to discern, via fecal metabolites, distinct characteristics between these two subtypes12,35. The recognition of specific metabolites intricately linked with distinct disease states not only underscores a promising avenue for early detection and patient stratification but also heralds the potential to explore the mechanistic underpinnings of IBD. De Preter et al.10, while assessing the variation in VOCs in patients with active CD and UC (according to the Harvey-Bradshaw Index and Ulcerative Colitis Disease Activity Index, respectively), noted that 1-propanol was significantly higher in active CD, whereas styrene levels significantly and positively correlated with the UC Disease Activity Index (UCDAI). In our study, we also identified differences in the activity pattern based on the type of IBD: elevated levels of octadecanoic acid were seen in individuals with active CD, whereas in UC, higher levels of both octadecanoic acid and hexadecanoic acid were detected, with disease activity identified by increased FC levels. Elevated levels of hexadecanoic acid or palmitic acid have been associated with inflammatory activity in various scenarios, such as metabolic dysfunction-associated fatty liver disease (MAFLD)42, astrocytes29, and particularly in intestinal cells, significantly impacting intestinal barrier integrity15. This alteration impairs paracellular permeability and cell-cell junctions and modifies cytokine expression profiles in intestinal epithelial cells14. Jansson et al.18 also observed higher levels of this metabolite, along with oleic acid, stearic acid, 6Z-, 9Z-, and 12Z-octadecatrienoic acid, linoleic acid, and arachidonic acid, in patients with ileal CD compared with colonic CD and healthy individuals, although these authors exclusively assessed CD patients. Our findings suggest that the elevation of palmitic acid may be directly related to the extent of lesion in UC patients, as active UC patients exhibited higher levels of this metabolite compared with those with lower inflammatory activity. In contrast to the findings in this study, where octadecanoic acid or stearic acid emerged as a differentiating biomarker between CD and UC, and for increased activity in the latter, Tefas et al.39 identified that not only hexadecanoic acid but also its three isomers (stearyl palmitoleate, palmitoleyl stearate, and oleyl palmitate) were higher in the serum of patients with colonic CD than in those with UC. Elevated fecal levels of stearic acid have also been observed in individuals with familial Mediterranean fever - an autoinflammatory disorder characterized by periodic attacks of acute inflammation and fever - as well as peptic ulceration22. This metabolite, along with heptanoic acid, constituted the most abundant evenand odd-numbered carbon SLCFAs in the colon lumen of a rat model of neonatal maternal separation. These SLCFAs promoted colonic muscle contraction and increased stool frequency in rats43. Collectively, these findings suggest that octadecanoic acid not only contributes to greater inflammatory activity but also plays a role in increased intestinal peristalsis, consequently stimulating diarrhea in UC patients. Similarly to hexadecanoic acid and octadecanoic acid, squalene was a metabolite found in higher concentrations in individuals with UC compared to those with CD. Squalene, an insoluble isoprenoid compound, represents one of the intermediates in the cholesterol biosynthesis pathway, serving as a precursor for hormones, bile acids, and vitamin D31. It also exhibits antioxidant, anti-inflammatory, and drug carrier effects21. However, some studies have highlighted negative associations with this metabolite27. Elevated plasma levels of squalene have been observed in women with cardiovascular diseases33, correlating with visceral obesity19, implying that squalene could serve as a biomarker for cardiovascular diseases30. Regarding intestinal cells, an intriguing study by Jiang et al.19 conducted on colon adenocarcinoma samples revealed that squalene synthase (an enzyme involved in squalene synthesis) accelerated colon cancer cell proliferation and promoted tumor growth. The inhibition of this enzyme, when combined with squalene epoxidase inhibition, resulted in a more pronounced suppressive effect on cell proliferation and tumor growth compared with single inhibition. In this context, the elevation of squalene, notably found in UC patients who are at a higher risk of developing colorectal cancer, might prove a valuable biomarker for the clinical monitoring of these individuals. This study does present some limitations, such as the convenience sampling method and the absence of microbiota composition analysis. Additionally, certain unidentified VOCs found in the study remain beyond the scope of discussion and associations with IBD clinical implications. However, the significance of investigating fecal metabolites becomes evident due to their potential for distinguishing CD from UC, monitoring disease activity, and evaluating clinical progression in these patients. Furthermore, the identification of these metabolites opens a broad spectrum of possibilities for pharmacological and nonpharmacological therapeutic targets. However, given the intricate relationship between microbiota and fecal metabolites, intertwined with living conditions, dietary habits, and past and present history, it is imperative to explore diverse populations, thus forming a more robust database to inform effective clinical practices. CONCLUSIONS In this context, distinctions in fecal metabolites were unearthed among patients with CD and UC, as well as those with heightened disease activity, as assessed by elevated FC levels, underscoring the potential of these metabolites in discriminating IBD subtypes and gauging disease severity. Collectively, these findings hold promise in informing therapeutic alternatives for IBD, including fecal microbiota transplantation and antioxidant therapy. However, several questions remain unanswered, given the presence of numerous unidentified compounds that might play significant roles in disease understanding. Consequently, further extensive investigations are imperative to shed light on these aspects in the future. Figure 1 Patterns of separation between Crohn’s disease (CD) and ulcerative colitis (UC) by partial least squares discriminant analysis (PLS-DA). The green area corresponds to patients with CD, and the pink area corresponds to patients with UC. Variables in projection (VIP) score (B). Perspectives This study shows that distinctions in fecal metabolites were unearthed among patients with CD and UC, as well as those with heightened disease activity, as assessed by elevated FC levels, underscoring the potential of these metabolites in discriminating IBD subtypes and gauging disease severity. Collectively, these findings hold promise in informing therapeutic alternatives for IBD, including fecal microbiota transplantation and antioxidant therapy. Central Message The etiology of IBD is unknown, but the interaction between genetics, immunological, and environmental factors, such as microbiota, contribute to its development. In this context, intestinal dysbiosis - the imbalance of the intestinal microbiota - is closely related to the pathogenesis and clinical complications of IBD; however, it is still not possible to distinguish whether this dysbiosis is a cause or a consequence of the disease. In recent years, studies have aimed to identify laboratory markers of IBD, with high sensitivity and specificity for noninvasive diagnosis and monitoring of these diseases. linkedin.com/company/revista-abcd facebook.com/Revista-ABCD-109005301640367 twitter.com/revista_abcd instagram.com/revistaabcd/ Financial source: None Editorial Support: National Council for Scientific and Technological Development (CNPq). How to cite this article: Lins LC, De-Meira JEC, Pereira CW, Crispim AC, Gischewski MDR, Lins-Neto MAF, et al. Fecal calprotectin and intestinal metabolites: what is their importance in the activity and differentiation of patients with inflammatory bowel diseases? ABCD Arq Bras Cir Dig. 2025;38e1870. https://doi.org/10.1590/0102-6720202500001e1870. REFERENCES 1 1 Ahmed I, Greenwood R, Costello B, Ratcliffe N, Probert CS. Investigation of faecal volatile organic metabolites as novel diagnostic biomarkers in inflammatory bowel disease. Aliment Pharmacol Ther. 2016;43(5):596-611. https://doi.org/10.1111/apt.13522 Ahmed I Greenwood R Costello B Ratcliffe N Probert CS. Investigation of faecal volatile organic metabolites as novel diagnostic biomarkers in inflammatory bowel disease Aliment Pharmacol Ther 2016 43 5 596 611 https://doi.org/10.1111/apt.13522 2 2 Alshehri D, Saadah O, Mosli M, Edris S, Alhindi R, Bahieldin A. Dysbiosis of gut microbiota in inflammatory bowel disease: current therapies and potential for microbiota-modulating therapeutic approaches. Bosn J Basic Med Sci. 2021;21(3):270-83. https://doi.org/10.17305/bjbms.2020.5016 Alshehri D Saadah O Mosli M Edris S Alhindi R Bahieldin A. Dysbiosis of gut microbiota in inflammatory bowel disease: current therapies and potential for microbiota-modulating therapeutic approaches Bosn J Basic Med Sci 2021 21 3 270 283 https://doi.org/10.17305/bjbms.2020.5016 3 3 Bauset C, Gisbert-Ferrándiz L, Cosín-Roger J. Metabolomics as a promising resource identifying potential biomarkers for inflammatory bowel disease. J Clin Med. 2021;10(4):622. https://doi.org/10.3390/jcm10040622 Bauset C Gisbert-Ferrándiz L Cosín-Roger J. Metabolomics as a promising resource identifying potential biomarkers for inflammatory bowel disease J Clin Med 2021 10 4 622 https://doi.org/10.3390/jcm10040622 4 4 Beckonert O, Keun HC, Ebbels TM, Bundy J, Holmes E, Lindon JC, et al. Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts. Nat Protoc. 2007;2(11):2692-703. https://doi.org/10.1038/nprot.2007.376 Beckonert O Keun HC Ebbels TM Bundy J Holmes E Lindon JC Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts Nat Protoc 2007 2 11 2692 2703 https://doi.org/10.1038/nprot.2007.376 5 5 Bjarnason I. The use of fecal calprotectin in inflammatory bowel disease. Gastroenterol Hepatol (N Y). 2017;13(1):53-6. PMID: 28420947. Bjarnason I. The use of fecal calprotectin in inflammatory bowel disease Gastroenterol Hepatol (N Y) 2017 13 1 53 56 28420947. 6 6 Bjerrum JT, Wang Y, Hao F, Coskun M, Ludwig C, Günther U, et al. Metabonomics of human fecal extracts characterize ulcerative colitis, Crohn’s disease and healthy individuals. Metabolomics. 2015;11:122-33. https://doi.org/10.1007/s11306-014-0677-3 Bjerrum JT Wang Y Hao F Coskun M Ludwig C Günther U Metabonomics of human fecal extracts characterize ulcerative colitis, Crohn’s disease and healthy individuals Metabolomics 2015 11 122 133 https://doi.org/10.1007/s11306-014-0677-3 7 7 Bryce C, Bucaj M. Fecal calprotectin for the evaluation of inflammatory bowel disease. Am Fam Physician. 2021;104(3):303-4. PMID: 34523882. Bryce C Bucaj M. Fecal calprotectin for the evaluation of inflammatory bowel disease Am Fam Physician 2021 104 3 303 304 34523882. 8 8 Carbery I, Burdge G, Clark T, Broglio G, Greer D, Alakkari A, et al. Impact on direct and indirect costs of switching patients with inflammatory bowel disease from intravenous to subcutaneous infliximab (CT-P13). BMJ Open Gastroenterol. 2023;10(1):e001105. https://doi.org/10.1136/bmjgast-2023-001105 Carbery I Burdge G Clark T Broglio G Greer D Alakkari A Impact on direct and indirect costs of switching patients with inflammatory bowel disease from intravenous to subcutaneous infliximab (CT-P13) BMJ Open Gastroenterol 2023 10 1 e001105 https://doi.org/10.1136/bmjgast-2023-001105 9 9 Clish CB. Metabolomics: an emerging but powerful tool for precision medicine. Cold Spring Harb Mol Case Stud. 2015;1(1):a000588. https://doi.org/10.1101/mcs.a000588 Clish CB. Metabolomics: an emerging but powerful tool for precision medicine Cold Spring Harb Mol Case Stud 2015 1 1 a000588 https://doi.org/10.1101/mcs.a000588 10 10 De Preter V, Machiels K, Joossens M, Arijs I, Matthys C, Vermeire S, et al. Faecal metabolite profiling identifies medium-chain fatty acids as discriminating compounds in IBD. Gut. 2015;64(3):447-58. https://doi.org/10.1136/gutjnl-2013-306423 De Preter V Machiels K Joossens M Arijs I Matthys C Vermeire S Faecal metabolite profiling identifies medium-chain fatty acids as discriminating compounds in IBD Gut 2015 64 3 447 458 https://doi.org/10.1136/gutjnl-2013-306423 11 11 Fiehn O, Kind T. Metabolite profiling in blood plasma. Methods Mol Biol. 2007;358:3-17. https://doi.org/10.1007/978-1-59745-244-1_1 Fiehn O Kind T. Metabolite profiling in blood plasma Methods Mol Biol 2007 358 3 17 https://doi.org/10.1007/978-1-59745-244-1_1 12 12 Franzosa EA, Sirota-Madi A, Avila-Pacheco J, Fornelos N, Haiser HJ, Reinker S, et al. Gut microbiome structure and metabolic activity in inflammatory bowel disease. Nat Microbiol. 2019;4(2):293-305. https://doi.org/10.1038/s41564-018-0306-4 Franzosa EA Sirota-Madi A Avila-Pacheco J Fornelos N Haiser HJ Reinker S Gut microbiome structure and metabolic activity in inflammatory bowel disease Nat Microbiol 2019 4 2 293 305 https://doi.org/10.1038/s41564-018-0306-4 13 13 Garner CE, Smith S, de Lacy Costello B, White P, Spencer R, Probert CSJ, et al. Volatile organic compounds from feces and their potential for diagnosis of gastrointestinal disease. FASEB J. 2007;21(8):1675-88. https://doi.org/10.1096/fj.06-6927com Garner CE Smith S de Lacy Costello B White P Spencer R Probert CSJ Volatile organic compounds from feces and their potential for diagnosis of gastrointestinal disease FASEB J 2007 21 8 1675 1688 https://doi.org/10.1096/fj.06-6927com 14 14 Ghezzal S, Postal BG, Quevrain E, Brot L, Seksik P, Leturque A, et al. Palmitic acid damages gut epithelium integrity and initiates inflammatory cytokine production. Biochim Biophys Acta Mol Cell Biol Lipids. 2020;1865(2):158530. https://doi.org/10.1016/j.bbalip.2019.158530 Ghezzal S Postal BG Quevrain E Brot L Seksik P Leturque A Palmitic acid damages gut epithelium integrity and initiates inflammatory cytokine production Biochim Biophys Acta Mol Cell Biol Lipids 2020 1865 2 158530 https://doi.org/10.1016/j.bbalip.2019.158530 15 15 Gori M, Altomare A, Cocca S, Solida E, Ribolsi M, Carotti S, et al. Palmitic acid affects intestinal epithelial barrier integrity and permeability in vitro. Antioxidants (Basel). 2020;9(5):417. https://doi.org/10.3390/antiox9050417 Gori M Altomare A Cocca S Solida E Ribolsi M Carotti S Palmitic acid affects intestinal epithelial barrier integrity and permeability in vitro Antioxidants (Basel) 2020 9 5 417 https://doi.org/10.3390/antiox9050417 16 16 Guan Q. A comprehensive review and update on the pathogenesis of inflammatory bowel disease. J Immunol Res. 2019;2019:7247238. https://doi.org/10.1155/2019/7247238 Guan Q. A comprehensive review and update on the pathogenesis of inflammatory bowel disease J Immunol Res 2019 2019 7247238 https://doi.org/10.1155/2019/7247238 17 17 Jacobs JP, Goudarzi M, Singh N, Tong M, McHardy IH, Ruegger P, et al. A disease-associated microbial and metabolomics state in relatives of pediatric inflammatory bowel disease patients. Cell Mol Gastroenterol Hepatol. 2016;2(6):750-66. https://doi.org/10.1016/j.jcmgh.2016.06.004 Jacobs JP Goudarzi M Singh N Tong M McHardy IH Ruegger P A disease-associated microbial and metabolomics state in relatives of pediatric inflammatory bowel disease patients Cell Mol Gastroenterol Hepatol 2016 2 6 750 766 https://doi.org/10.1016/j.jcmgh.2016.06.004 18 18 Jansson J, Willing B, Lucio M, Fekete A, Dicksved J, Halfvarson J, et al. Metabolomics reveals metabolic biomarkers of Crohn’s disease. PLoS One. 2009;4(7):e6386. https://doi.org/10.1371/journal.pone.0006386 Jansson J Willing B Lucio M Fekete A Dicksved J Halfvarson J Metabolomics reveals metabolic biomarkers of Crohn’s disease PLoS One 2009 4 7 e6386 https://doi.org/10.1371/journal.pone.0006386 19 19 Jiang H, Tang E, Chen Y, Liu H, Zhao Y, Lin M, et al. Squalene synthase predicts poor prognosis in stage I-III colon adenocarcinoma and synergizes squalene epoxidase to promote tumor progression. Cancer Sci. 2022;113(3):971-85. https://doi.org/10.1111/cas.15248 Jiang H Tang E Chen Y Liu H Zhao Y Lin M Squalene synthase predicts poor prognosis in stage I-III colon adenocarcinoma and synergizes squalene epoxidase to promote tumor progression Cancer Sci 2022 113 3 971 985 https://doi.org/10.1111/cas.15248 20 20 Kamlage B, Maldonado SG, Bethan B, Peter E, Schmitz O, Liebenberg V, et al. Quality markers addressing preanalytical variations of blood and plasma processing identified by broad and targeted metabolite profiling. Clin Chem. 2014;60(2):399-412. https://doi.org/10.1373/clinchem.2013.211979 Kamlage B Maldonado SG Bethan B Peter E Schmitz O Liebenberg V Quality markers addressing preanalytical variations of blood and plasma processing identified by broad and targeted metabolite profiling Clin Chem 2014 60 2 399 412 https://doi.org/10.1373/clinchem.2013.211979 21 21 Kim SK, Karadeniz F. Biological importance and applications of squalene and squalane. Adv Food Nutr Res. 2012;65:223-33. https://doi.org/10.1016/B978-0-12-416003-3.00014-7 Kim SK Karadeniz F. Biological importance and applications of squalene and squalane Adv Food Nutr Res 2012 65 223 233 https://doi.org/10.1016/B978-0-12-416003-3.00014-7 22 22 Ktsoyan ZA, Beloborodova NV, Sedrakyan AM, Osipov GA, Khachatryan ZA, Kelly D, et al. Profiles of microbial fatty acids in the human metabolome are disease-specific. Front Microbiol. 2011;1:148. https://doi.org/10.3389/fmicb.2010.00148 Ktsoyan ZA Beloborodova NV Sedrakyan AM Osipov GA Khachatryan ZA Kelly D Profiles of microbial fatty acids in the human metabolome are disease-specific Front Microbiol 2011 1 148 https://doi.org/10.3389/fmicb.2010.00148 23 23 Le Gall G, Noor SO, Ridgway K, Scovell L, Jamieson C, Johnson IT, et al. Metabolomics of fecal extracts detects altered metabolic activity of gut microbiota in ulcerative colitis and irritable bowel syndrome. J Proteome Res. 2011;10(9):4208-18. https://doi.org/10.1021/pr2003598 Le Gall G Noor SO Ridgway K Scovell L Jamieson C Johnson IT Metabolomics of fecal extracts detects altered metabolic activity of gut microbiota in ulcerative colitis and irritable bowel syndrome J Proteome Res 2011 10 9 4208 4218 https://doi.org/10.1021/pr2003598 24 24 Lee BL, Rout M, Mandal R, Wishart DS. Automated identification and quantification of metabolites in human fecal extracts by nuclear magnetic resonance spectroscopy. Magn Reson Chem. 2023;61(12):705-17. https://doi.org/10.1002/mrc.5372 Lee BL Rout M Mandal R Wishart DS. Automated identification and quantification of metabolites in human fecal extracts by nuclear magnetic resonance spectroscopy Magn Reson Chem 2023 61 12 705 717 https://doi.org/10.1002/mrc.5372 25 25 Marchesi JR, Holmes E, Khan F, Kochhar S, Scanlan P, Shanahan F, et al. Rapid and noninvasive metabonomic characterization of inflammatory bowel disease. J Proteome Res. 2007;6(2):546-51. https://doi.org/10.1021/pr060470d Marchesi JR Holmes E Khan F Kochhar S Scanlan P Shanahan F Rapid and noninvasive metabonomic characterization of inflammatory bowel disease J Proteome Res 2007 6 2 546 551 https://doi.org/10.1021/pr060470d 26 26 Mendonça CM, Correa Neto IJF, Rolim AS, Robles L. Inflammatory bowel diseases: characteristics, evolution, and quality of life. Arq Bras Cir Dig. 2022;35:e1653. https://doi.org/10.1590/0102-672020210002e1653 Mendonça CM Correa IJF Neto Rolim AS Robles L. Inflammatory bowel diseases: characteristics, evolution, and quality of life Arq Bras Cir Dig 2022 35 e1653 https://doi.org/10.1590/0102-672020210002e1653 27 27 Micera M, Botto A, Geddo F, Antoniotti S, Bertea CM, Levi R, et al. Squalene: more than a step toward sterols. Antioxidants (Basel). 2020;9(8):688. https://doi.org/10.3390/antiox9080688 Micera M Botto A Geddo F Antoniotti S Bertea CM Levi R Squalene: more than a step toward sterols Antioxidants (Basel) 2020 9 8 688 https://doi.org/10.3390/antiox9080688 28 28 Moura FA, Andrade KQ, Santos JCF, Araújo ORP, Goulart MOF. Antioxidant therapy for treatment of inflammatory bowel disease: does it work? Redox Biol. 2015;6:617-39. https://doi.org/10.1016/j.redox.2015.10.006 Moura FA Andrade KQ Santos JCF Araújo ORP Goulart MOF. Antioxidant therapy for treatment of inflammatory bowel disease: does it work? Redox Biol 2015 6 617 639 https://doi.org/10.1016/j.redox.2015.10.006 29 29 Ortiz-Rodriguez A, Acaz-Fonseca E, Boya P, Arevalo MA, Garcia-Segura LM. Lipotoxic effects of palmitic acid on astrocytes are associated with autophagy impairment. Mol Neurobiol. 2019;56(3):1665-80. https://doi.org/10.1007/s12035-018-1183-9 Ortiz-Rodriguez A Acaz-Fonseca E Boya P Arevalo MA Garcia-Segura LM. Lipotoxic effects of palmitic acid on astrocytes are associated with autophagy impairment Mol Neurobiol 2019 56 3 1665 1680 https://doi.org/10.1007/s12035-018-1183-9 30 30 Peltola P, Pihlajamäki J, Koutnikova H, Ruotsalainen E, Salmenniemi U, Vauhkonen I, et al. Visceral obesity is associated with high levels of serum squalene. Obesity (Silver Spring). 2006;14(7):1155-63. https://doi.org/10.1038/oby.2006.132 Peltola P Pihlajamäki J Koutnikova H Ruotsalainen E Salmenniemi U Vauhkonen I Visceral obesity is associated with high levels of serum squalene Obesity (Silver Spring) 2006 14 7 1155 1163 https://doi.org/10.1038/oby.2006.132 31 31 Picón DF, Skouta R. Unveiling the therapeutic potential of squalene synthase: deciphering its biochemical mechanism, disease implications, and intriguing ties to ferroptosis. Cancers (Basel). 2023;15(14):3731. https://doi.org/10.3390/cancers15143731 Picón DF Skouta R. Unveiling the therapeutic potential of squalene synthase: deciphering its biochemical mechanism, disease implications, and intriguing ties to ferroptosis Cancers (Basel) 2023 15 14 3731 https://doi.org/10.3390/cancers15143731 32 32 Pillai N, Dusheiko M, Burnand B, Pittet V. A systematic review of cost-effectiveness studies comparing conventional, biological and surgical interventions for inflammatory bowel disease. PLoS One. 2017;12(10):e0185500. https://doi.org/10.1371/journal.pone.0185500 Pillai N Dusheiko M Burnand B Pittet V. A systematic review of cost-effectiveness studies comparing conventional, biological and surgical interventions for inflammatory bowel disease PLoS One 2017 12 10 e0185500 https://doi.org/10.1371/journal.pone.0185500 33 33 Rajaratnam RA, Gylling H, Miettinen TA. Independent association of serum squalene and noncholesterol sterols with coronary artery disease in postmenopausal women. J Am Coll Cardiol. 2000;35(5):1185-91. https://doi.org/10.1016/s0735-1097(00)00527-1 Rajaratnam RA Gylling H Miettinen TA. Independent association of serum squalene and noncholesterol sterols with coronary artery disease in postmenopausal women J Am Coll Cardiol 2000 35 5 1185 1191 https://doi.org/10.1016/s0735-1097(00)00527-1 34 34 Rokkas T, Portincasa P, Koutroubakis IE. Fecal calprotectin in assessing inflammatory bowel disease endoscopic activity: a diagnostic accuracy meta-analysis. J Gastrointestin Liver Dis. 2018;27(3):299-306. https://doi.org/10.15403/jgld.2014.1121.273.pti Rokkas T Portincasa P Koutroubakis IE. Fecal calprotectin in assessing inflammatory bowel disease endoscopic activity: a diagnostic accuracy meta-analysis J Gastrointestin Liver Dis 2018 27 3 299 306 https://doi.org/10.15403/jgld.2014.1121.273.pti 35 35 Santoru ML, Piras C, Murgia A, Palmas V, Camboni T, Liggi S, et al. Cross sectional evaluation of the gut-microbiome metabolome axis in an Italian cohort of IBD patients. Sci Rep. 2017;7(1):9523. https://doi.org/10.1038/s41598-017-10034-5 Santoru ML Piras C Murgia A Palmas V Camboni T Liggi S Cross sectional evaluation of the gut-microbiome metabolome axis in an Italian cohort of IBD patients Sci Rep 2017 7 1 9523 https://doi.org/10.1038/s41598-017-10034-5 36 36 Sobrado LF, Mori FNC, Facanali CBG, Camargo MGM, Nahas SC, Sobrado CW. Risk factors for early postoperative complications in acute colitis in the era of biologic therapy. Arq Bras Cir Dig. 2023;36:e1770. https://doi.org/10.1590/0102-672020230052e1770 Sobrado LF Mori FNC Facanali CBG Camargo MGM Nahas SC Sobrado CW. Risk factors for early postoperative complications in acute colitis in the era of biologic therapy Arq Bras Cir Dig 2023 36 e1770 https://doi.org/10.1590/0102-672020230052e1770 37 37 Takaishi H, Matsuki T, Nakazawa A, Takada T, Kado S, Asahara T, et al. Imbalance in intestinal microflora constitution could be involved in the pathogenesis of inflammatory bowel disease. Int J Med Microbiol. 2008;298(5-6):463-72. https://doi.org/10.1016/j.ijmm.2007.07.016 Takaishi H Matsuki T Nakazawa A Takada T Kado S Asahara T Imbalance in intestinal microflora constitution could be involved in the pathogenesis of inflammatory bowel disease Int J Med Microbiol 2008 298 5-6 463 472 https://doi.org/10.1016/j.ijmm.2007.07.016 38 38 Tamboli CP, Neut C, Desreumaux P, Colombel JF. Dysbiosis in inflammatory bowel disease. Gut. 2004;53(1):1-4. https://doi.org/10.1136/gut.53.1.1 Tamboli CP Neut C Desreumaux P Colombel JF. Dysbiosis in inflammatory bowel disease Gut 2004 53 1 1 4 https://doi.org/10.1136/gut.53.1.1 39 39 Tefas C, Ciobanu L, Tanţău M, Moraru C, Socaciu C. The potential of metabolic and lipid profiling in inflammatory bowel diseases: a pilot study. Bosn J Basic Med Sci. 2020;20(2):262-70. https://doi.org/10.17305/bjbms.2019.4235 Tefas C Ciobanu L Tanţău M Moraru C Socaciu C. The potential of metabolic and lipid profiling in inflammatory bowel diseases: a pilot study Bosn J Basic Med Sci 2020 20 2 262 270 https://doi.org/10.17305/bjbms.2019.4235 40 40 Wang Y, Juan L, Peng J, Wang T, Zang T, Wang Y. Explore potential disease related metabolites based on latent factor model. BMC Genomics. 2022;23(Suppl 1):269. https://doi.org/10.1186/s12864-022-08504-w Wang Y Juan L Peng J Wang T Zang T Wang Y. Explore potential disease related metabolites based on latent factor model BMC Genomics 2022 23 Suppl 1 269 https://doi.org/10.1186/s12864-022-08504-w 41 41 Want EJ, Wilson ID, Gika H, Theodoridis G, Plumb RS, Shockcor J, et al. Global metabolic profiling procedures for urine using UPLC-MS. Nat Protoc. 2010;5(6):1005-18. https://doi.org/10.1038/nprot.2010.50 Want EJ Wilson ID Gika H Theodoridis G Plumb RS Shockcor J Global metabolic profiling procedures for urine using UPLC-MS Nat Protoc 2010 5 6 1005 1018 https://doi.org/10.1038/nprot.2010.50 42 42 Yamada S, Kamada N, Amiya T, Nakamoto N, Nakaoka T, Kimura M, et al. Gut microbiota-mediated generation of saturated fatty acids elicits inflammation in the liver in murine high-fat diet-induced steatohepatitis. BMC Gastroenterol. 2017;17(1):136. https://doi.org/10.1186/s12876-017-0689-3 Yamada S Kamada N Amiya T Nakamoto N Nakaoka T Kimura M Gut microbiota-mediated generation of saturated fatty acids elicits inflammation in the liver in murine high-fat diet-induced steatohepatitis BMC Gastroenterol 2017 17 1 136 https://doi.org/10.1186/s12876-017-0689-3 43 43 Zhao L, Huang Y, Lu L, Yang W, Huang T, Lin Z, et al. Saturated long-chain fatty acid-producing bacteria contribute to enhanced colonic motility in rats. Microbiome. 2018;6(1):107. https://doi.org/10.1186/s40168-018-0492-6 Zhao L Huang Y Lu L Yang W Huang T Lin Z Saturated long-chain fatty acid-producing bacteria contribute to enhanced colonic motility in rats Microbiome 2018 6 1 107 https://doi.org/10.1186/s40168-018-0492-6