Introduction

Inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative colitis (UC), are chronic advanced immune-mediated inflammatory diseases of unknown etiologies [1, 2]. There is still no unified “gold standard” for the diagnosis of IBDs; the diagnosis is usually made on the basis of data sets which include medical history, clinical manifestations, typical endoscopic views and histological signs, therefore, between 5% and 15% of cases of IBDs do not meet the strict criteria for either UC or CD, and in 14% of patients diagnosed with both UC and CD, the diagnosis can be changed over time [3, 4].

Aims & Methods

Research objective is as follows: To develop a diagnostic method for the IBDs including differential diagnosis, using a combination of indicators, such as fecal, hematological, serological, fatty acids' (FA) levels of erythrocyte (RBC) membranes and blood serum (BS).
A total of 91 patients (average age 37.7 ± 12.1 years old) with IBDs and 53 patients of the comparison group (43.3 ± 11.7 years old) have been examined. The group of patients with IBDs have included patients with ulcerative colitis (UC) – 50 persons and with Crohn’s disease (CD) – 41 persons. In the examined patients, the red blood parameters and biochemical parameters have been studied by standard methods, as for the level of fecal calprotectin, it has been researched by the enzyme-linked immunosorbent assay (ELISA), and the levels of fatty acids of erythrocyte membranes and blood serum have been investigated by the gas chromatography–mass spectrometry (GC-MS) (Agilent 7000B, USA).

Results

In a comparative analysis of patient groups using statistical analysis, a composite set of indicators has been found to be the most significant in distinguishing the patients with acute UC from comparison group individuals: hemoglobin levels (p<0.00001), sedimentation rate (p<0.00001), ferritin levels (p<0.00001), fecal calprotectin (p<0.00001), and oleic acid content (p=0.0031), polyunsaturated fatty acids in erythrocyte membranes (p<0.05), arachidonic acid in erythrocyte membranes and blood serum (p<0.05), and serum PUFAs (p=0.0018) and n6/n3 PUFA ratio (p=0.0005). Diagnostic models have been developed using machine learning methods using data analysis libraries of the Python programming language. The first model, which includes a combination of the above indicators, allows to perform the calculation of the diagnostic index Y _D1 to differentiate healthy individuals from patients with IBDs (AUC 0.957, sensitivity 0.974, specificity 0.941). The second diagnostic model for distinguishing patients with acute ulcerative colitis from patients with active Crohn's disease (AUC 0.887, sensitivity 0.993, specificity 0.840) have considered the levels of hemoglobin, hematocrit, mean corpuscular volume of erythrocytes (p=0.007), mean cell hemoglobin concentration and content in the RBCs (p<0.05), ESR (p<0.05), C-reactive protein (p=0.002), ferritin (p=0.014), fibrinogen (p<0.0001), fecal calprotectin (p=0.005), and erythrocyte level of oleic acid (p<0.05).

Conclusion

The diagnostic models that have been created include fecal, serum markers of inflammation, as well as levels of fatty acids pathogenetically associated with the activity of IBDs, which ensures their high diagnostic accuracy. The non-invasive nature, an availability of indicators, and a convenient algorithm allow us to optimize and expedite the diagnosis of the IBDs.

References

1. Kaplan G.G., Ng S.C. Understanding and preventing the global increase of inflammatory bowel disease. Gastroenterology. 2017 Feb; 152(2):313-321.e2. DOI: 10.1053/j.gastro.2016.10.020.
2. Maaser C., Sturm A., Vavricka S.R., Kucharzik T., Fiorino G., Annese V., et al. European Crohn’s and Colitis Organisation [ECCO] and the European Society of Gastrointestinal and Abdominal Radiology [ESGAR]. EC-CO-ESGAR Guideline for Diagnostic Assessment in IBD Part 1: Initial diagnosis, monitoring of known IBD, detection of complications. J Crohns Colitis. 2019;13(2):144– 64. DOI: 10.1093/ecco-jcc/jjy113
3. Abraham B.P., Mehta S., El-Serag H.B. Natural history of pediatric-onset inflammatory bowel disease: a systematic review. J Clin Gastroenterol. 2012;46(7):581–9. DOI: 10.1097/MCG.0b013e318247c32f
4. Henriksen M., Jahnsen J., Lygren I., Sauar J., Schulz T., Stray N., et al. Change of diagnosis during the first five years after onset of inflammatory bowel disease: results of a prospective follow-up study (the IBSEN Study). Scand J Gastroenterol. 2006;41(9):1037–43. DOI: 10.1080/00365520600554527.

Introduction

Unclear etiology of IBD, the frequent similarity of their clinical and endoscopic manifestations, lack of a gold standard in diagnosing diseases, which complicates the choice of targeted therapy, urgently require improving the efficiency of currently available diagnostic technologies [1]. Significant efforts are being made to search for biomarkers that can be used to identify patients at high risk of IBD relapse who need more intensive monitoring and therapy in the early stages of these diseases, in remission to prevent long-term irreversible structural damage [2].

Aims & Methods

The purpose of the research was to study the levels of fatty acids (FAs) in red blood cells (rbc) membranes and blood serum in patients with IBD examined under follow-up control as possible predictors of disease exacerbation.
During our study, 44 patients with IBD have been monitored, in the course of that period, the average age was equal to 36.3±5.2. The first examination has been performed in remission of the diseases, the second one 6 to 12 months after the first step, and it has been done in the acute stage of IBD. The composition of the FAs has been studied using gas chromatography-mass spectrometry (Agilent 7000B (USA).

Results

Predictors of exacerbation progress in patients with IBD have been found to be reduced levels of such FAs as C20:5 n-3 (p=0.002), C18:3 n-3 (p=0.039), C20:2 n-6 (p=0.002), C16:2 n-6 (p=0.006), C22: 4 n-6 (p=0.008), and on the contrary, increased content of FA C18:0 (p=0.005), SFAs/USFAs (p=0.039), SFAs/PUFAs (p=0.03) in rbc membranes and FA C20:0 in blood serum (p=0.008). The diagnostic model created was able to predict the exacerbation progress with a sensitivity of 87.2%, specificity of 91.3% (AUC 0.936), incorporating the above-described fatty acids with respect to their degree of contribution. We have found correlations of rbc C18:0 with haemoglobin level (r=-0.344, p<0.001), erythrocyte sedimentation rate (r=0.480, p=0.012), fibrinogen level (r=0.352, p=0.004); rbc C18:3n-3 - with level of C-reactive protein (r=-0.472, p=0.023), fibrinogen (r=-0.420, p=0.022), faecal calprotectin (r=-0.391, p=0.044). The content of rbc C16:2n-6 was directly correlated with the level of fecal calprotectin (r=0.407, p=0.028), ratio of SFAs/USFAs was depending on the number of leukocytes (r=0.411, p=0.046). The function of elevated levels of SFAs as predictors of exacerbation of IBD is probably determined by the fact that they potentially regulate inflammatory reactions by acting on the pathways of the PPAR-γ and retinoid X receptors (RXR) [3]. Reduced levels of C18:3n-3 as a predictor of exacerbation may be related to the ability of this FA to reduce the activity of ionised calcium-binding adaptor molecule 1-positive macrophages [4] and to reduce the expression of proinflammatory genes IL-8, COX2 and inducible nitric oxide synthase to reduce intestinal inflammation [5]. The significance of C20:5n-3 for prediction of the active stage of IBD is due to the fact that it inhibits the inflammatory pathways of transforming growth factor beta-1/epidermal growth factor receptor P-EGFR and NF-κB, and it also modulates redox balance and mitigates the progression of UC [6]. Reduced levels of several omega-6 PUFAs may be related to their increased consumption during elongation, desaturation, including in the arachidonic cascade associated with the cynthesis of proinflammatory cytokines [7].

Conclusion

The use of fatty acids of rbc membranes and blood serum is promising and pathogenetically justified, namely as biomarkers for predicting the exacerbation progress in patients with IBD.

References

1. Seyedian SS, Nokhostin F, Malamir MD. A review of the diagnosis, prevention, and treatment methods of inflammatory bowel disease. J Med Life. 2019 Apr-Jun;12(2):113-122. doi: 10.25122/jml-2018-0075.
2. Tibble J.A., Sigthorsson G., Bridger S.et al. Surrogate markers of intestinal inflammation are predictive of relapse in patients with inflammatory bowel disease. Gastroenterology. 2000; 119: 15–22. doi:10.1053/gast.2000.8523
3. Mozaffarian D. Trans fatty acids effects on systemic inflammation and endothelial function. Atheroscler Suppl (2006) 7(2):29–32. doi: 10.1016/ j.atherosclerosissup.2006.04.007
4. Kim J, Ahn M, Choi Y. et al. Alpha-linolenic acid alleviates dextran sulfate sodium-induced ulcerative colitis in mice. Inflammation (2020) 43(5):1876–83. doi: 10.1007/s10753-020-01260-7
5. Reifen R, Karlinsky A, Stark AH. et al. A-linolenic acid (Ala) is an anti-inflammatory agent in inflammatory bowel disease. J Nutr Biochem (2015) 26 (12):1632–40. doi: 10.1016/j.jnutbio.2015.08.006
6. El Mahdy RN, Nader MA, Helal MG. et al. Eicosapentaenoic acid mitigates ulcerative colitis-induced by acetic acid through modulation of nf-Kb and tgf-B/egfr signaling pathways. Life Sci (2023) 327:121820. doi: 10.1016/j.lfs.2023.121820
7. Ajabnoor SM, Thorpe G, Abdelhamid A, Hooper L. Long-term effects of increasing omega-3, omega-6 and total polyunsaturated fats on inflammatory bowel disease and markers of inflammation: a systematic review and meta-analysis of randomized controlled trials. Eur J Nutr. 2021 Aug;60(5):2293-2316. doi: 10.1007/s00394-020-02413-y.

Introduction

Inflammatory bowel diseases (IBD) are chronic, heterogeneous, and inflammatory conditions mainly affecting the gastrointestinal tract. Currently, endoscopy is the gold standard test for assessing mucosal activity and healing in clinical practice; however, it is a costly, time-consuming, invasive, and uncomfortable procedure for patients. There is, therefore, a need for sensitive, specific, fast and non-invasive biomarkers for the diagnosis of IBD.

Aims & Methods

Label-free quantification by nanoscale liquid chromatography coupled to tandem mass spectrometry (nLC MS/MS) was performed to profile the urinary and serum proteomes of 100 patients with newly diagnosed IBD, before starting any treatment [50 patients with Crohn´s disease (CD) and 50 patients with ulcerative colitis (UC)] and 50 healthy controls (HC). Data Mining and Pattern Recognition techniques were used to identify hidden relationships that are not detectable by using the classical and most commonly used lineal classifiers; thus, identifying potential markers able to discriminate the studied cohorts. Finally, we applied Ingenuity Pathway Analysis (IPA) to analyze the pathways and functions of differentially expressed proteins.

Results

Serum proteomics results revealed 45 differentially expressed proteins in the comparison between UC and HC groups, 32 proteins significantly expressed in CD versus HC, and 12 proteins in CD compared with UC. In urine samples, 110 proteins were significantly changed in UC versus HC, 50 proteins were differentially expressed between CD and HC, and a total of 31 proteins were significantly changed in CD compared with UC patients. Receiver operating characteristic curve (ROC) analysis found multiple proteins with a high area under the curve values, up to 0.94, indicating that these serum and urine proteins are of value as new non-invasive diagnostic classifiers of IBD patients. For each study comparative, the 5 most significant classifiers were selected. IPA revealed multiple signaling pathways, including prothrombin activation, acute phase response signaling, complement and coagulation system and liver X receptor/retinoid X receptor (LXR/RXR) activation, altered in IBD patients compared to HC (p-value < 0.05).

Conclusion

Our findings indicate that analysis of the urine and serum proteome using nLC MS/MS is a feasible approach for biomarker discovery. We identified several serum and urine proteins that could serve as new non-invasive markers for the diagnosis of IBD patients after further validation. After bioinformatic analyses, we found multiple proteins that may play important roles in the pathogenesis of IBD.

Introduction

Apelin, an adipokine secreted from visceral fat, has been associated with endothelial cells dysfunction‐related chronic inflammatory diseases. Decreased serum apelin levels have been found in patients with cardiovascular disease (CVD). Higher apelin levels have been measured in mesenteric adipose tissue and inflamed colonic epithelium of patients with Inflammatory Bowel Disease (IBD), but data on the role of serum apelin levels in IBD patients are lacking.

Aims & Methods

We aimed to a) compare serum apelin levels between 104 consecutive IBD patients and 104 age and sex matched healthy controls (blood donors), b) to evaluate the correlation between serum apelin levels with demographics, disease characteristics, treatment and disease course.
Serum apelin-13 levels were measured using ELISA (Wuhan Fine Biological Technology Co., Ltd). Statistical analysis was performed with SPSS 24 (SPSSInc. Chicago, IL, USA).

Results

Mean apelin levels were significantly higher in IBD patients compared to their matched healthy controls (1996.29 ± 1592.96pg/ml vs 1552.99 ± 809.64, p=0.012). Among the examined parameters only smoking was found independently associated with serum apelin levels in IBD patients (p<0.001).
IBD patients with CVD (25) had lower but not significant different serum apelin levels compared to patients without CVD (79) (1743.01 ± 1116.26pg/dl vs 2465.09 ± 2006.51pg/ml, p=0.122). In a subgroup analysis of patients without CVD (79 IBD patients) significantly elevated serum apelin levels were found in patients with extended UC, history of immunomodulator use and absence of musculoskeletal EIMs (2539,69 ± 1969.29pg/ml vs 1164.45 ± 818.89pg/ml, p=0.044, 2317.69 ± 1867.99pg/ml vs 1451.40 ± 1024.20pg/ml, p=0.043 and 2340.58 ± 1850.16pg/ml vs 1392.09 ± 1054.99pg/ml, p=0.027, respectively).
Finally, a moderate positive correlation was found among UC extent and serum apelin levels in patients without CVD (r=0.525, p=0.004). Νegative correlations were found among age at study entry and serum apelin levels in both IBD patients without CVD and all IBD patients (r=-0.263, p=0.001 and r=-0.214, p=0.002, respectively) and between immunomodulator use, history of musculoskeletal EIMs and serum apelin levels in patients without CVD (r=-0.26, p=0.021 and r=-0.336, p=0.005, respectively).

Conclusion

The circulating serum apelin levels are significantly increased in IBD patients compared to matched healthy controls. Increased apelin levels are independently associated with smoking status and disease extend (in UC). IBD patients with musculoskeletal manifestations have decreased serum apelin levels compared to IBD patients without musculoskeletal manifestations. Further investigation with larger studies on the role of serum apelin in IBD is needed.

Disclosure

None

Introduction

A differential diagnosis of inflammatory bowel diseases (IBD) and irritable bowel syndrome (IBS) is one of the unresolved problems in modern gastroenterology. The absence of clear criteria for IBD detection leads to delay in the diagnostic process for months and years.

Aims & Methods

The aim of the study was to identify clinical and laboratory markers that could distinguish between IBD and IBS with a subsequent development of an artificial neural network (ANN).
We retrospectively evaluated the clinical symptoms, anamnesis data and laboratory tests of patients with verified exacerbation of Crohn`s disease (CD) and ulcerative colitis (UC), and IBS. We found parameters that could distinguish IBD and IBS and then CD and UC, and used them to create the ANN.

Results

Of the 1006 examined, 32% were patients with IBS and 68% with IBD. Patients with IBS were significantly more likely to have a tendency to constipation (34.8% compared with 5.0% in IBD patients), loose stool up to 2 times (26.9% compared with 17.0%), the absence of extraintestinal (EIMs) (94.1% compared to 66.3%) and perianal manifestations (PAM) (96.0% compared to 83.5%) (p<0.001). The patients with IBD had diarrheal syndrome ≥ 5 times a day more frequently (40.0% and 6.2% in patients with IBS, respectively), blood admixture (65.3% and 6.8%, respectively) and higher level of fecal calprotectin (p<0.001). All these data were used to create and train the ANN on general and test (15% of all cases) dataset. Multilayer perceptron showed the best results in IBD detection: the sensitivity was 95.8% and the specificity was 90.2%.
To create the ANN for IBD differentiation, we analyzed medical histories of 350 patients (51%) with UC and 333 (49%) with CD. Statistical analysis in the group with UC significantly more often had severe diarrheal syndrome (≥ 5 times a day) (48.3% compared to 42.3% among the patients with CD), blood in the stool (87.1% compared to 79.0%, respectively), the absence of EIMs (73,4% compared to 58,9%, respectively) and PAMs (97,4% compared to 58,9%, respectively) (p<0,001). Patients with CD compared with UC were significantly more likely to have constipation (7.2% compared to 2.9%, respectively) (p=0.002), increased stool up to 2 times (21,6% compared to 12,6%, respectively), subfebrile temperature (25.8% compared to 16.9%, respectively) (p=0.004) and surgical intervention on the small and large intestines in the anamnesis (20.1% in compared to 1.4%, respectively) (p<0.001). The clinical and anamnestic parameters identified in our study were used to create and train the ANN. The best results were shown by a linear set with 5 input neurons: the accuracy of detecting CD was 78.7% and UC - 75.4%, which shows a moderate ability of the ANN to differentiate UC and CD.

Conclusion

As a result of the study, we created the ANNs with high sensitivity and specificity in the differentiation of organic and functional intestinal diseases and with moderate accuracy in IBD differentiation. The developed ANN can help clinicians in the diagnosis of IBD and IBS, but further work for improving its accuracy and the possibility of implementation into clinical practice is required.

Disclosure

All authors have declared no conflict of interest.