Application of alpha-zeenol for screening risk of Crohn disease

Abstract

The invention provides application of alpha-ZAL in screening the risk of Crohn disease, and belongs to the technical field of biochemical detection. According to the present invention, the endocrine disrupter alpha-zeenol is adopted as the marker for screening the risk of the Crohn disease, the high performance liquid chromatography-tandem mass spectrometry detection technology is adopted to detect the endocrine disrupter, and the endocrine disrupter is prepared into the kit for the risk analysis of the Crohn disease.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical detection, and specifically to the use of α - zearalenol for screening the risk of Crohn's disease. Background Art

[0002] Crohn's disease is a type of inflammatory bowel disease (IBD), a chronic non - specific granulomatous inflammatory disease of the gastrointestinal tract, characterized by segmental and non - uniform transmural infection. The cause is unknown, it can occur in any part of the digestive tract, and has a cyclic course of repeated attacks and remissions. The clinical manifestations are repeated abdominal pain, diarrhea, accompanied by abdominal masses, intestinal fistulas, anal lesions, and often accompanied by extra - intestinal lesions, such as arthritis, iridocyclitis, etc. The diagnosis and treatment of CD are both difficult, with a long course of disease and a long time for confirmation. It is easy to cause abdominal complications such as intestinal stenosis and fistulas, depression, anxiety, cancer and other diseases, and may require several surgeries in a lifetime, having a serious negative impact on the quality of life and psychology of patients. Moreover, in recent years, with the improvement of people's living standards and the change of diet structure, the incidence of CD shows an increasing trend year by year. Therefore, it is urgent to conduct early diagnosis or pre - examination of Crohn's disease to improve the diagnosis rate, shorten the confirmation time, and achieve the purpose of early diagnosis and early regular treatment.

[0003] Currently, the diagnosis of Crohn's disease mainly relies on the comprehensive diagnosis of clinical symptoms, endoscopic pathology and imaging examinations. However, the above - mentioned indicators all start to increase after the occurrence of Crohn's disease, and the confirmation period is relatively long, and they cannot be used as screening markers for early - onset risk factors. In the prior art, fecal calprotectin is also monitored to judge the intestinal inflammation situation, but calprotectin can only reflect intestinal inflammation and cannot diagnose Crohn's disease. Therefore, this method is more used for detecting the intestinal inflammation situation of confirmed patients and is difficult to be used for screening of risk factors. Anti - Saccharomyces cerevisiae antibodies (ASCA) or anti - neutrophil cytoplasmic antibodies (ANCA) are considered to be valuable for the diagnosis of Crohn's disease, but their sensitivity and specificity are significantly lower in the Chinese disease population than in the European and American populations, so they are not used as routine examination items for CD. Detection of Crohn's disease - related genes, such as ATG16L1, IL23R, IRGM and NOD2, can also be used as indicators for risk screening, but there are few institutions with the ability to perform these detections, and it is still difficult to popularize. Summary of the Invention

[0004] The purpose of the present invention is to provide a screening method for Crohn's disease. In view of the deficiencies in the prior art, the present invention provides an endocrine disruptor for screening Crohn's disease and its application. In the present invention, the zearalenone endocrine disruptor is used as a marker for screening Crohn's disease, and the above - mentioned endocrine disruptor is detected by high - performance liquid chromatography - tandem mass spectrometry detection technology, and is prepared into a kit for risk analysis of Crohn's disease.

[0005] To achieve the above - mentioned purpose, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a composition for screening the risk of Crohn's disease, the composition comprising a solution of α - zeaxanthol.

[0007] Preferably, the solution is an acetonitrile solution of α - zeaxanthol.

[0008] In a second aspect, the present invention provides a kit for screening the risk of Crohn's disease, the kit comprising a urine sample pretreatment reagent, an eluent, a reconstitution solution, a diluent, and a mixed standard working solution, wherein the mixed standard working solution is a solution containing α - zeaxanthol.

[0009] Preferably, the mixed standard working solution is an acetonitrile solution of α - zeaxanthol.

[0010] Additionally preferably, the urine sample pretreatment reagent comprises a buffer, β - glucuronidase / arylsulfatase, and vitamin C.

[0011] Furthermore, the buffer is an acetic acid - sodium acetate buffer, wherein the acetic acid - sodium acetate buffer is prepared by mixing a 0.2M aqueous acetic acid solution and a 0.2M aqueous sodium acetate solution in a volume ratio of 1:6.

[0012] Preferably, the A phase of the eluent is an aqueous solution of 0.1% (v / v) formic acid, and the B phase is an acetonitrile solution of 0.1% (v / v) formic acid.

[0013] Preferably, the reconstitution solution is an aqueous acetonitrile solution of 0.1% formic acid, wherein the volume ratio of acetonitrile to water is 1:9.

[0014] Preferably, the diluent is an aqueous solution containing 2 g / mL urea and 1 g / mL sodium chloride, for diluting the urine matrix.

[0015] In a third aspect, the present invention provides the use of α - zeaxanthol for preparing a composition for screening the risk of Crohn's disease.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] In the present invention, based on the fact that environmental toxins account for a relatively high proportion in the pathogenesis of Crohn's disease, the zearalenone endocrine disruptor is used as a biomarker for screening Crohn's disease, and it is prepared into a kit for early diagnosis of Crohn's disease. Starting from the perspective of etiology, the present invention provides a biomarker and a diagnostic kit for monitoring the pathogenic factors leading to the disease before the onset of the disease, which has important clinical value for early screening of the risk of Crohn's disease.

[0018] In the present invention, there is further provided a kit for early diagnosis of Crohn's disease based on endocrine disruptor indicators. This kit has the advantages of simple sample acquisition, low detection limit, good repeatability, high screening sensitivity and specificity, etc., and overcomes the limitations of the one-sidedness of current clinical diagnostic indicators for Crohn's disease, the lag in the diagnosis stage, the complexity of detection methods, and the difficulty in obtaining samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the standard curve drawn by using the standard product working solution when screening samples with the kit of the present invention;

[0020] Figure 2 It is the product ion mass spectrum of the detection object α-ZAL;

[0021] Figure 3 It is the Logistic regression analysis of the α-ZAL urine residue level and the onset of Crohn's disease;

[0022] Figure 4 It is the detection results of α-ZAL in urine samples of the normal control group and the Crohn's disease group;

[0023] Figure 5 It is the ROC curve for the early screening performance detection of α-ZAL. DETAILED DESCRIPTION OF THE INVENTION

[0024] In the present invention, there is first provided the use of the endocrine disruptor zearalenone for screening the risk of Crohn's disease.

[0025] Among them, the Crohn's disease is caused by chemical toxins in the environment.

[0026] In the present invention, there is also provided the application of the above endocrine disruptor in the preparation of reagents or kits for screening, and the Crohn's disease is caused by chemical toxins in the environment.

[0027] In the present invention, there is also provided a kit for screening, including: urine sample pretreatment reagent combination, eluent, mixed standard product working solution, diluent, eluent / activation solution, eluent / activation solution, reconstitution solution.

[0028] Furthermore, the urine sample pretreatment reagent combination includes acetic acid-sodium acetate buffer solution, β-glucuronidase / arylsulfatase, and vitamin C. Among them, the acetic acid-sodium acetate buffer solution is composed of 0.2M acetic acid aqueous solution and 0.2M sodium acetate aqueous solution mixed in a volume ratio of 1:6; the dosage ratio of the acetic acid-sodium acetate buffer solution, β-glucuronidase / arylsulfatase, and vitamin C is 2 mL:10 μL:5 mg.

[0029] The A phase of the eluent is an aqueous solution of 0.1% (v / v) formic acid, and the B phase is an acetonitrile solution of 0.1% (v / v) formic acid;

[0030] The standard working solution is an acetonitrile solution containing endocrine disruptors. The endocrine disruptor is α-zearalenol (α-ZAL, a specific metabolite of zearalenone), and the concentration is 1 μg / mL.

[0031] The diluent is an aqueous solution containing 2 g / mL urea and 1 g / mL sodium chloride.

[0032] The eluent / activating solution is water.

[0033] The eluent / activating solution is acetonitrile.

[0034] The reconstitution solution is an aqueous acetonitrile solution of 0.1% (v / v) formic acid, and the volume ratio of acetonitrile to water is 1:9.

[0035] The present invention also provides a method for using the above kit, which specifically includes the following steps:

[0036] (1) Collect samples from clinically suspected Crohn's disease patients, and use the urine sample pretreatment reagent combination to process the urine samples;

[0037] (2) Use the mixed standard working solution in the kit to prepare a mixed standard solution with a concentration gradient;

[0038] (3) Inject the prepared test samples and standard solutions into the LC-MS / MS mass spectrometer for detection;

[0039] (4) According to the detection results, draw a standard curve of the mixed standard solution to screen the risk of Crohn's disease.

[0040] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0041] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "upper", "lower", "inner", "outer", "front end", "rear end", "both ends", "one end", "the other end", etc. is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be construed as a limitation of the present invention. In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance.

[0042] In the description of the present invention, it should be noted that unless otherwise clearly specified and defined, terms such as "installation", "provided with", "connection", etc. should be understood in a broad sense. For example, "connection" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two components. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0043] Example 1: Preparation of a kit for screening

[0044] Components and preparation of the kit for screening:

[0045] Urine sample pretreatment reagent combination: acetic acid - sodium acetate buffer, β - glucuronidase / arylsulfatase, vitamin C; wherein, the acetic acid - sodium acetate buffer is prepared by mixing 0.2M acetic acid aqueous solution and 0.2M sodium acetate aqueous solution in a volume ratio of 1:6.

[0046] Standard working solution: Using acetonitrile as the solvent, the concentration of the solute α - ZAL is 1 μg / mL.

[0047] Diluent: Urine matrix diluent, specifically an aqueous solution containing 2 g / mL urea and 1 g / mL sodium chloride.

[0048] Washing solution / activation solution: Water

[0049] Elution solution / activation solution: Acetonitrile

[0050] Reconstitution solution: An acetonitrile aqueous solution of 0.1% formic acid, and the volume ratio of acetonitrile to water is 1:9.

[0051] Example 2: Method for screening with the kit

[0052] (1) Treatment of the solution to be tested:

[0053] Take 2 mL of urine sample, first filter it with a 0.22 μm filter membrane, add acetic acid - sodium acetate buffer, adjust the pH value to 5.4, then add 10 μL of β - glucuronidase / arylsulfatase to specifically degrade the substances to be tested that are glucuronidated or sulfonated in the urine sample, then add 5 mg of vitamin C, and incubate overnight at room temperature. For the enzymatically digested sample, perform solid - phase extraction using an SPE column (C18ENVI, 0.25 g), and elute the extract with 2 mL of acetonitrile - water, dry it with nitrogen, and re - dissolve it with 100 μL of an acetonitrile aqueous solution of 0.1% formic acid to obtain the solution to be tested.

[0054] (2) Plotting of the standard curve:

[0055] Appropriately transfer the standard solution (1 μg / ml) from the standard working solution, and dilute it with the urine matrix diluent to 100 ng / mL, 50 ng / mL, 20 ng / mL, 10 ng / mL, 5 ng / mL, 1 ng / mL, and 0.5 ng / mL. Then, perform the detection under the chromatographic conditions and mass spectrometry conditions described below. After the standard curve is plotted, the actual samples can be quantified using the standard curve. The standard curve of the standard is as Figure 1 shown, where Y = 13927X + 10984, R = 0.9993, and the detection limit is 0.1 ng / mL.

[0056] The described chromatographic conditions are as follows:

[0057] Mobile phase: Phase A: Aqueous solution of 0.1% (v / v) formic acid, Phase B: Acetonitrile solution of 0.1% (v / v) formic acid;

[0058] Chromatographic column: ACQUITY UPLC BEH C18 chromatographic column;

[0059] Elution method: Gradient elution, and the gradient elution program table is shown in Table 1.

[0060] Table 1. Gradient program

[0061] Time (min) Phase A (%) Phase B (%) 2 80 20 8 0 100 9 0 100 10 80 20 12 Stop

[0062] The described mass spectrometry conditions are as follows:

[0063]

[0064] Table 2. MRM parameters

[0065]

[0066] In Table 2, α-ZAL: α-zearalenol; Precursor ion: The characteristic ion generated by the endocrine disruptor in the first-stage mass spectrometry (MS) mode, also called the parent ion; The product ion is the characteristic fragment ion generated by the parent ion of the endocrine disruptor after collision with an inert gas (such as argon) in the second-stage (MS / MS) mass spectrometry mode, also called the daughter ion.

[0067] The product ion mass spectrum of α-ZAL is as Figure 2 shown.

[0068] Draw the standard curve according to the detection results, and the drawing result is as Figure 3As shown. It can be seen from the figure that there is a significant positive correlation between α-ZAL and the onset of Crohn's disease. In this example, the trimethylsilylation derivatization combined with GC-MS method was used as the control group, and the kit prepared in the present invention was used as the experimental group to detect α-ZAL with a theoretical concentration of 100 ng / mL, so as to investigate the accuracy of the detection result of α-ZAL by the kit prepared in the present invention. The investigation results are shown in Table 3.

[0069] Table 3. Recovery rates of α-ZAL under different detection methods

[0070] α-ZAL Concentration (ng / mL) α-ZAL Recovery Rate (%) Control Group 87.2 87 Experimental Group 96.3 96

[0071] Among them, the recovery rate is the ratio of the average value of the measured concentration to the theoretical concentration. It can be seen from the above table that compared with the trimethylsilylation derivatization combined with GC-MS method for detecting α-ZAL in the prior art, the kit prepared in the present invention has a higher accuracy rate when detecting α-ZAL.

[0072] Example 3: Verification of the screening effect of the detection result on the risk of Crohn's disease

[0073] To verify whether the above detection method and the level of α-ZAL in the body can be used for the early risk screening of Crohn's disease, in this example, urine samples (10 mL) of 32 normal adolescent volunteers and urine samples (10 mL) of 30 patients diagnosed with Crohn's disease were collected. The urine samples were pretreated, and then mass spectrometry was performed on the machine and data analysis was carried out. The processing method and detection conditions of the samples to be tested were exactly the same as those in Example 2.

[0074] The detection results of α-ZAL in the urine samples of the normal control group and the Crohn's disease group are shown in Figure 4 .

[0075] Data processing method in this Example 1: The screening ability of each index can be evaluated by calculating the area under the ROC curve (AUC). Use Graphpad to draw the difference graph of toxin content, and all statistical analyses were completed in the statistical calculation environment of SPSS (IBM, V26) software.

[0076] Figure 5 For the ROC curve of α-ZAL, ROC curve analysis was performed on the detection results. The results showed that the area under the curve (AUC) of α-ZAL in urine for screening the risk of Crohn's disease was 92%, and it showed significant statistical significance (P < 0.0001). The above results overall illustrate the residual situation of this endocrine disruptor metabolite α-ZAL in the urine of normal people and patients with Crohn's disease, and it can be used as a screening index for the risk of Crohn's disease. It can also be seen from the funnel plot of the prediction performance that the method of the present invention has very high specificity and sensitivity.

[0077] Where the present invention is not elaborated, it is the well-known technology to those skilled in the art.

[0078] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail with reference to the embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified and equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A composition for screening the risk of Crohn's disease, characterized in that, The composition comprises a solution of α - zeaxanthol.

2. The composition for screening the risk of Crohn's disease according to claim 1, wherein The solution is an acetonitrile solution of α - zeaxanthol.

3. A kit for screening the risk of Crohn's disease, characterized in that, The kit comprises a urine sample pretreatment reagent, an eluent, a reconstitution solution, a diluent, and a mixed standard working solution, wherein the mixed standard working solution is a solution containing α - zeaxanthol.

4. The kit for screening the risk of Crohn's disease according to claim 3, wherein The mixed standard working solution is an acetonitrile solution of α - zeaxanthol.

5. The kit for screening the risk of Crohn's disease according to claim 3, characterized in that, The urine sample pretreatment reagent comprises a buffer, β - glucuronidase / arylsulfatase, and vitamin C.

6. The kit for screening the risk of Crohn's disease according to claim 5, characterized in that, The buffer is an acetic acid - sodium acetate buffer, wherein the acetic acid - sodium acetate buffer is prepared by mixing a 0.2 M aqueous acetic acid solution and a 0.2 M aqueous sodium acetate solution in a volume ratio of 1:

6.

7. The kit for screening the risk of Crohn's disease according to claim 3, wherein The A phase of the eluent is an aqueous solution of 0.1% (v / v) formic acid, and the B phase is an acetonitrile solution of 0.1% (v / v) formic acid.

8. The kit for screening the risk of Crohn's disease according to claim 3, wherein, The reconstitution solution is an acetonitrile aqueous solution of 0.1% formic acid, wherein the volume ratio of acetonitrile to water is 1:

9.

9. The kit for screening the risk of Crohn's disease according to claim 3, wherein The diluent is an aqueous solution containing 2 g / mL urea and 1 g / mL sodium chloride, and is used to dilute the urine matrix.

10. Use of α - zeaxanthol for the preparation of a composition for screening the risk of Crohn's disease.

Images