Department of Oral Biochemistry, Academic Centre for Dentistry Amsterdam, University of Amsterdam and VU University Amsterdam, Amsterdam, The Netherlands.
Department of Pediatric Gastroenterology, Emma Children's Hospital, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands.
FASEB J. 2024 May 15;38(9):e23627. doi: 10.1096/fj.202302190R.
Colonoscopy is the gold standard for diagnosing inflammatory bowel disease (IBD). However, this invasive procedure has a high burden for pediatric patients. Previous research has shown elevated fecal amino acid concentrations in children with IBD versus controls. We hypothesized that this finding could result from increased proteolytic activity. Therefore, the aim of this study was to investigate whether fecal protease-based profiling was able to discriminate between IBD and controls. Protease activity was measured in fecal samples from patients with IBD (Crohn's disease (CD) n = 19; ulcerative colitis (UC) n = 19) and non-IBD controls (n = 19) using a fluorescence resonance energy transfer (FRET)-peptide library. Receiver operating characteristic (ROC) curve analysis was used to determine the diagnostic value of each FRET-peptide substrate. Screening the FRET-peptide library revealed an increased total proteolytic activity (TPA), as well as degradation of specific FRET-peptides specifically in fecal samples from IBD patients. Based on level of significance (p < .001) and ROC curve analysis (AUC > 0.85), the fluorogenic substrates W-W, A-A, a-a, F-h, and H-y showed diagnostic potential for CD. The substrates W-W, a-a, T-t, G-v, and H-y showed diagnostic potential for UC based on significance (p < .001) and ROC analysis (AUC > 0.90). None of the FRET-peptide substrates used was able to differentiate between protease activity in fecal samples from CD versus UC. This study showed an increased fecal proteolytic activity in children with newly diagnosed, treatment-naïve, IBD. This could lead to the development of novel, noninvasive biomarkers for screening and diagnostic purposes.
结肠镜检查是诊断炎症性肠病（IBD）的金标准。然而，这种有创的程序对儿科患者来说负担很重。先前的研究表明，IBD 患儿的粪便氨基酸浓度升高与对照组相比。我们假设这种发现可能是由于蛋白酶活性增加所致。因此，本研究旨在探讨粪便蛋白酶谱分析是否能够区分 IBD 和对照组。使用荧光共振能量转移（FRET）肽库测量来自 IBD 患者（克罗恩病（CD）n=19；溃疡性结肠炎（UC）n=19）和非 IBD 对照组（n=19）的粪便样本中的蛋白酶活性。使用接收者操作特征（ROC）曲线分析来确定每个 FRET-肽底物的诊断价值。筛选 FRET-肽文库显示 IBD 患者粪便中总蛋白水解酶活性（TPA）增加，以及特定 FRET-肽的降解增加。基于显著性（p<0.001）和 ROC 曲线分析（AUC>0.85），荧光底物 W-W、A-A、a-a、F-h 和 H-y 对 CD 具有诊断潜力。基于显著性（p<0.001）和 ROC 分析（AUC>0.90），底物 W-W、a-a、T-t、G-v 和 H-y 对 UC 具有诊断潜力。用于区分 CD 和 UC 粪便中蛋白酶活性的任何 FRET-肽底物都无法区分。本研究显示，新诊断、未经治疗的 IBD 儿童的粪便蛋白酶活性增加。这可能导致用于筛查和诊断目的的新型非侵入性生物标志物的发展。
