Fecal biomarker for diagnosing inflammatory bowel disease and application thereof

Abstract

The invention discloses an excrement biomarker for diagnosing inflammatory bowel diseases and application of the excrement biomarker. The excrement biomarker is adrenoic acid. It is found through experiments that adrenoic acid is up-regulated in an IBD patient excrement sample for the first time, and it is found through detection that expression of adrenoic acid is up-regulated in the IBD patient excrement sample; further, compared with other inflammation activity indexes such as cadherin, the adrenoic acid has better diagnosis sensitivity and specificity through ROC curve analysis. A new diagnostic marker is provided for clinical diagnosis of IBD, adrenoic acid is used as a faeces biomarker for IBD diagnosis, is used for diagnosis of inflammatory bowel diseases, and has the advantages of high detection accuracy, high specificity and sensitivity, convenience, rapidness, safety, noninvasive property and the like; the method is of great significance in judging the activity state of IBD, adjusting diagnosis, treatment and management of IBD patients and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of inflammatory bowel disease detection, and in particular to a fecal biomarker for diagnosing inflammatory bowel disease and an application thereof. Background Art

[0002] Inflammatory bowel disease (IBD) is a type of chronic gastrointestinal inflammatory disease with an unknown etiology, mainly including ulcerative colitis (UC), Crohn's disease (CD) and indeterminate colitis (IC). Patients often experience repeated attacks during the long course of the disease, forming a "relapse-remission-relapse" cycle, which can lead to fibrosis and decreased function of the intestinal wall, thus significantly affecting the quality of life. At present, the medical burden of inflammatory bowel disease has become an important public health challenge.

[0003] Timely determination of the activity status of IBD can help reduce the surgical rate and mortality rate. At present, the pathogenesis and key molecules of IBD have not been fully clarified. The current diagnosis mainly relies on endoscopic and pathological examinations, but due to their invasiveness and subjectivity, as well as their time-consuming, labor-intensive, and non-repeatable operation, their widespread application is limited. At present, the assessment of IBD disease activity mainly relies on: 1) laboratory indicators, including plasma inflammatory markers (such as CRP, ESR, ALB, etc.); 2) fecal inflammatory indicators, mainly fecal calprotectin; 3) disease activity scores, including endoscopic scores (such as SESCD, CDEIS, and Rutgeerts scores) and clinical scores (such as CDAI and Mayo scores). However, the existing plasma inflammatory markers are not specifically for gastrointestinal inflammation, and the test results of fecal calprotectin are not stable enough, which affects the accuracy of the assessment. Endoscopic or clinical comprehensive evaluation may be affected by subjective factors, and there is a bias in the assessment for patients with mild symptoms or endoscopic manifestations.

[0004] The activity assessment of IBD is crucial for guiding subsequent medication and judging the prognosis of patients. Objectively reflecting intestinal mucosal inflammation through highly sensitive and specific biomarkers, thereby judging the activity of IBD, is particularly critical for optimizing diagnosis and treatment and managing patients. Metabolites are extracted from fecal samples as markers. Feces are easy to obtain, simple to operate, and do not require blood sampling. Patients are more likely to accept it and it is more suitable for a wide range of people. Fecal markers have the advantages of being non-invasive, simple, fast, and repeatable in a short period of time. Therefore, the study of new markers based on feces (such as metabolites) has broad clinical application prospects and is of great significance for judging the activity of IBD, adjusting the diagnosis and treatment of IBD patients, and managing IBD patients. Summary of the invention

[0005] The purpose of the present invention is to provide a fecal biomarker for diagnosing inflammatory bowel disease and its application in view of the problems existing in the prior art. The fecal biomarker has stable chemical properties and is easy to detect. It is used for diagnosing inflammatory bowel disease and has the advantages of high detection accuracy, high specificity and sensitivity, convenience, speed, safety and non-invasiveness.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: A fecal biomarker for diagnosing inflammatory bowel disease, wherein the fecal biomarker is adrenal acid.

[0007] Furthermore, the fecal biomarker for diagnosing inflammatory bowel disease is used in the preparation of a product for diagnosing inflammatory bowel disease.

[0008] Furthermore, the products for diagnosing inflammatory bowel disease include detection reagents and test kits.

[0009] Furthermore, the inflammatory bowel disease includes ulcerative colitis and Crohn's disease.

[0010] The present invention provides the use of adrenal acid as a fecal biomarker in the preparation of a detection reagent or a kit for diagnosing or screening inflammatory bowel disease / monitoring the activity of inflammatory bowel disease.

[0011] On the other hand, the present invention also provides the use of a reagent for detecting fecal biomarkers in the preparation of a product for diagnosing inflammatory bowel disease, comprising: a) identifying a biomarker in a fecal sample of a subject, wherein the biomarker is adrenal acid; and b) comparing the biomarker with a reference, wherein the difference in the biomarker compared with the reference is used to detect inflammatory bowel disease.

[0012] Furthermore, the expression level of the biomarker is upregulated compared to a reference, which is a healthy population.

[0013] Furthermore, the above-mentioned reagents for detecting fecal biomarkers are used in the preparation of products for diagnosing inflammatory bowel disease, wherein the reagents include reagents for detecting adrenal acid using mass spectrometry, high performance liquid chromatography, ultra-high performance liquid chromatography-tandem mass spectrometry, and enzyme-linked immunosorbent assay.

[0014] Furthermore, methods for detecting the expression level of biomarkers in stool samples include, but are not limited to, mass spectrometry, high performance liquid chromatography (HPLC), ultra-high performance liquid chromatography-tandem mass spectrometry, and enzyme-linked immunosorbent assay (ELISA).

[0015] The beneficial effects of the present invention are: The present invention provides a new fecal biomarker for the diagnosis of inflammatory bowel disease. The present invention discloses adrenal acid as a fecal biomarker for the diagnosis of IBD for the first time, and discovers for the first time through experiments that adrenal acid is upregulated in fecal samples of IBD patients, suggesting that adrenal acid may have important clinical value in the disease activity and prognosis evaluation of IBD; it is found that adrenal acid is upregulated in fecal samples of IBD patients; further analysis of the ROC curve shows that adrenal acid has better diagnostic sensitivity and specificity than other inflammatory activity indicators such as cadherin; the area under the ROC curve of the adrenal acid marker in cynomolgus monkey IBD and IBD patients is much larger than that of CRP and fecal calprotectin activity indicators. The present invention provides a new diagnostic marker for the clinical diagnosis of IBD, which has important clinical significance.

[0016] The present invention uses adrenic acid as a fecal biomarker for the diagnosis of IBD, which is used for the diagnosis of inflammatory bowel disease, and has the advantages of high detection accuracy, high specificity and sensitivity, convenience, speed, safety and non-invasiveness, etc., and is of great significance for judging the activity state of IBD, adjusting the diagnosis and treatment of IBD patients, and management. The core metabolite disclosed in the present invention, adrenic acid, has important innovation and applicability as an IBD marker with its stable chemical properties and easy detection characteristics, and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Comparison of the expression levels of adrenaline in the IBD group and the normal group in Example 1; Figure 2 Comparison of the expression levels of adrenaline in the IBD group and the normal group in Example 2; Figure 3 ROC curve analysis of adrenal acid, fecal calprotectin, and CRP in cynomolgus monkey IBD; Figure 4 ROC curve analysis of adrenal acid, fecal calprotectin, and CRP in IBD patients. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with specific examples so that those skilled in the art can more clearly understand the present invention. In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0019] Example 1 Screening for markers associated with IBD 1. Sample collection Feces were collected from 17 IBD cynomolgus monkeys and 10 healthy cynomolgus monkeys. All the above specimens were obtained with the approval of the Animal Ethics Committee.

[0020] The inclusion criteria were based on the consensus opinion on the diagnosis and treatment of inflammatory bowel disease published by the Inflammatory Bowel Disease Group of the Gastroenterology Branch of the Chinese Medical Association (2018, Beijing). The exclusion criteria were obvious other intestinal diseases of the digestive system; a history of autoimmune diseases and malignant tumors. The inclusion criteria for healthy controls were no obvious digestive system intestinal diseases; no history of malignant tumors of the digestive system and other systems; no history of autoimmune diseases.

[0021] 2. Preparation and quality analysis of fecal samples Fecal samples were immediately stored in a -80°C ultra-low temperature freezer after collection. Before measurement, approximately 5 mg of each lyophilized sample was weighed and transferred to a new 1.5 mL PCR tube. Then 25 μL of ultrapure water was added, the zirconium oxide beads were homogenized for 3 minutes, and 120 μL of methanol-containing internal standard was added to extract metabolites. The samples were homogenized for another 3 minutes and then centrifuged at 18,000 g for 20 minutes. 20 μL of the supernatant was transferred to a 96-well plate. Subsequently, the plate was moved to an Eppendorf epMotion workstation. In the workstation environment, ice-cold methanol solution containing internal standard was added (total 120 μL) and vigorously vortexed for 5 minutes. Next, the plate was placed at 4°C for centrifugation (speed 4000 g, time 30 minutes), after which the plate was returned to the workstation. 20 μL of freshly prepared derivatization reagent was added to each well, and the plate was sealed and placed at 30°C for 60 minutes for derivatization reaction. Subsequently, the sample was diluted by adding 330 μL of 50% methanol solution in an ice bath and then centrifuged again at 4°C (speed 4000g, time 30 minutes). Next, the supernatant (total amount of 135 μL) was aspirated and transferred to a new 96-well plate, and 10 μL of internal standard was added to each well. The derivatized standard stock solution was added to the left well of the plate after serial dilution. Finally, the plate was sealed and LC-MS analysis was performed on a liquid chromatography-mass spectrometer (ACQUITY UPLC-Xevo TQ-S, Waters Corp., Milford, MA, USA). [The instrument conditions of UPLC-MS / MS were as follows: UPLC: chromatographic column: ACQUITYUPLC BEH C18 1.7μM VanGuard pre-column (2.1×5 mm) and ACQUITY UPLC BEH C181.7μM analytical column (2.1×100 mm); column temperature (℃): 40; sample manager temperature (℃): 10; mobile phase: A = water (0.1% formic acid), B = acetonitrile:IPA (70:30); elution conditions: 0-1 min (5% B), 1-5 min (5-30% B), 5-9 min (30-50% B), 9-11 min (50-78% B), 11-13.5 min (78-95% B), 13.5-14 min (95-100% B), 14-16 min (100% B), 16-16.1 min (100-5% B), 16.1-18 min (5% B); flow rate (mL / min): 0.40; injection volume (μL): 5.0.MASS SPECTROMETER: capillary voltage (Kv): 1.5 (ESI+), 2.0 (ESI-); source temperature (℃): 150; desolvation temperature (℃): 550; desolvation gas flow (L / Hr): 1000. The raw data files generated by UPLC-MS / MS were processed using MassLynx software. Peak integration, calibration and quantitative analysis of adrenic acid were performed.

[0022] 3. Results UPLC-MS / MS results showed that the expression level of adrenaline in patients with Crohn's disease and ulcerative colitis was significantly higher than that in normal monkeys ( Figure 1 ), suggesting that adrenal acid can be used as a possible personalized indicator in the diagnosis of inflammatory bowel disease.

[0023] Example 2 Screening for markers associated with IBD 1. Sample collection Feces were collected from 15 patients with IBD and 10 healthy people, and the patients gave informed consent. The acquisition of all the above specimens was approved by the ethics committee.

[0024] The inclusion criteria for patients with Crohn's disease and ulcerative colitis were based on the consensus opinion on the diagnosis and treatment of inflammatory bowel disease published by the Inflammatory Bowel Disease Group of the Chinese Society of Gastroenterology (2018, Beijing). Exclusion criteria were obvious other intestinal diseases of the digestive system; history of autoimmune diseases and malignant tumors; and failure to sign informed consent. The inclusion criteria for healthy controls were no obvious intestinal diseases of the digestive system; no history of malignant tumors of the digestive system and other systems; and no history of autoimmune diseases.

[0025] 2. Adrenaline acid test in stool samples Fecal samples were immediately stored in a -80°C ultra-low temperature freezer after collection. Before measurement, approximately 5 mg of each lyophilized sample was weighed and transferred to a new 1.5 mL PCR tube. Then 25 μL of ultrapure water was added, the zirconium oxide beads were homogenized for 3 minutes, and 120 μL of methanol-containing internal standard was added to extract metabolites. The samples were homogenized for another 3 minutes and then centrifuged at 18,000 g for 20 minutes. 20 μL of the supernatant was transferred to a 96-well plate. Subsequently, the plate was moved to an Eppendorf epMotion workstation. In the workstation environment, ice-cold methanol solution containing internal standard was added (total 120 μL) and vigorously vortexed for 5 minutes. Next, the plate was placed at 4°C for centrifugation (speed 4000 g, time 30 minutes), after which the plate was returned to the workstation. 20 μL of freshly prepared derivatization reagent was added to each well, and the plate was sealed and placed at 30°C for 60 minutes for derivatization reaction. Subsequently, 330 μL of 50% methanol solution in an ice bath was added to dilute the sample, and then centrifuged again at 4°C (speed 4000 g, time 30 minutes). Next, the supernatant (total amount of 135 μL) was aspirated and transferred to a new 96-well plate, and 10 μL of internal standard was added to each well. The gradient diluted derivatized standard stock solution was added to the left well of the plate. Finally, the plate was sealed and LC-MS analysis was performed on a liquid chromatography-mass spectrometer (ACQUITY UPLC-Xevo TQ-S, Waters Corp., Milford, MA, USA) (the instrument conditions of UPLC-MS / MS were the same as in Example 1). The raw data files generated by UPLC-MS / MS were processed using MassLynx software. Peak integration, calibration and quantitative analysis of adrenic acid were performed.

[0026] 3. Results The colorimetric results showed that the expression level of adrenal acid in patients with Crohn's disease and ulcerative colitis was significantly higher than that in normal people ( Figure 2 ), suggesting that adrenal acid can be used as a possible personalized indicator in the diagnosis of inflammatory bowel disease.

[0027] Example 3 Analysis of the sensitivity and specificity of adrenal acid for marking IBD The receiver operator characteristic curve (ROC curve) was used to analyze the effectiveness of adrenal acid in disease diagnosis and to divide the optimal threshold score. The ROC curve is a curve drawn with sensitivity as the ordinate and 1-specificity as the abscissa. It can accurately reflect the specificity and sensitivity of the analysis method. The more convex the ROC curve is near the upper left corner, the greater the diagnostic value. The area under the curve (AUC) can evaluate the accuracy of the diagnostic test. The area under the ROC curve ranges from 0.5 to 1. When it is 0.5, it means that the test has no diagnostic value. When the area is between 0.5-0.7, it has a lower accuracy. When the area is between 0.7-0.9, it means that it has a certain accuracy. When the area is >0.9, it has a higher accuracy. The ROC curve also helps to select the best diagnostic cutoff value and compare the recognition ability of different diagnostic test reports on diseases. It is currently a common method for determining the cutoff value of diagnostic tests.

[0028] At the same time, in order to enhance the comparison, this experiment also analyzed the area under the ROC curve of the activity indicators such as CRP and fecal calprotectin that have been disclosed in the prior art. The results are as follows Figure 3 As shown in the figure: The area under the ROC curve of adrenal acid markers in cynomolgus monkey IBD is much larger than that of CRP and fecal calprotectin activity indicators, indicating that adrenal acid can be used as a personalized indicator for the diagnosis of animal inflammatory bowel disease. The area under the ROC curve of adrenal acid markers in IBD patients is much larger than that of CRP and fecal calprotectin activity indicators ( Figure 4 ), indicating that adrenal acid can be used as a personalized indicator for the diagnosis of inflammatory bowel disease. The ROC analysis results based on sensitivity and specificity showed that adrenal acid has good IBD marker properties. Example 4

[0029] The use of a reagent for detecting fecal biomarkers in the preparation of a product for diagnosing inflammatory bowel disease comprises: a) identifying a biomarker in a fecal sample of a subject, wherein the biomarker is adrenic acid; and b) comparing the biomarker with a reference, wherein the difference in the biomarker compared with the reference is used to detect inflammatory bowel disease / detect disease activity of inflammatory bowel disease. Compared with the reference, the expression level of the biomarker is upregulated. The reference is a healthy population. The inflammatory bowel disease includes ulcerative colitis and Crohn's disease. The reagent includes a reagent for detecting adrenic acid by mass spectrometry, high performance liquid chromatography, ultra-high performance liquid chromatography-tandem mass spectrometry, and enzyme-linked immunosorbent assay. Methods for detecting the expression level of biomarkers in fecal samples may include, but are not limited to, mass spectrometry, high performance liquid chromatography (HPLC), ultra-high performance liquid chromatography-tandem mass spectrometry, and enzyme-linked immunosorbent assay (ELISA).

Claims

1. A fecal biomarker for the diagnosis of inflammatory bowel disease, characterized in that: The fecal biomarker is adrenal acid.

2. Use of the fecal biomarker for diagnosing inflammatory bowel disease as claimed in claim 1 in the preparation of a product for diagnosing inflammatory bowel disease.

3. The use according to claim 2, characterized in that: The products for diagnosing inflammatory bowel disease include detection reagents and test kits.

4. The use according to claim 3, characterized in that: The inflammatory bowel disease includes ulcerative colitis and Crohn's disease.

5. Use of a reagent for detecting fecal biomarkers in the preparation of a product for diagnosing inflammatory bowel disease, characterized in that: include: a) identifying a biomarker in a stool sample of a subject, wherein the biomarker is adrenaline; and b) comparing the biomarker to a reference, wherein the difference in the biomarker compared to the reference is used to detect inflammatory bowel disease.

6. The use according to claim 5, characterized in that: Biomarker expression levels were upregulated compared to the reference.

7. The use according to claim 5, characterized in that: The reference is a healthy population.

8. The use according to any one of claims 5 to 7, characterized in that: The reagents include reagents for detecting adrenal acid by mass spectrometry, high performance liquid chromatography, ultra-high performance liquid chromatography-tandem mass spectrometry, and enzyme-linked immunosorbent assay.

9. The use according to any one of claims 5 to 7, characterized in that: Methods for detecting the expression level of biomarkers in stool samples include, but are not limited to, mass spectrometry, high performance liquid chromatography, ultra-high performance liquid chromatography-tandem mass spectrometry, and enzyme-linked immunosorbent assay.

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