Application of 2-Hydroxy-2-methylbutyric acid in screening for cardiac cancer

Abstract

The present invention belongs to the field of biomedicine technology, and specifically discloses a marker for screening for cardia cancer. The marker for screening for cardia cancer provided by the present invention is 2-Hydroxy-2-methylbutyric acid, and the detection reagent of the marker can be used to prepare a cardia cancer screening product. By detecting the expression level of 2-Hydroxy-2-methylbutyric acid in human serum, cardia cancer can be effectively detected, which can be used for large-scale screening of asymptomatic people in areas with a high incidence of cardia cancer, which is conducive to screening and early detection of asymptomatic high-risk people.

Description

[0001] This application is a divisional application of the invention patent with application number CN 202110717684.7, application date 2021-06-28, and name “A marker and detection kit for screening of cardiac cancer”. Technical Field

[0002] The invention belongs to the field of medical biotechnology, and specifically discloses a marker for cardiac cancer screening and application thereof. Background Art

[0003] With the development of society and changes in the environment, cardia cancer has become a type of malignant tumor that seriously threatens human life, health and safety. Cardia cancer refers to cancer that occurs in the anatomical part of the cardia. It is generally believed in China that it is located within 2 cm below the esophageal-gastric junction, also known as cardia adenocarcinoma. Studies have found that in areas with a high incidence of esophageal cancer, there is also a high incidence of cardia cancer. This phenomenon suggests that there may be strong carcinogenic factors in the local living environment. These carcinogenic factors act on the upper gastrointestinal mucosa for a long time, and through some similar mechanisms, they have led to a high incidence of esophageal cancer and a high incidence of cardia cancer. Traditionally, "asymptomatic people in high-incidence areas, over 40 years old, male, smoking, drinking, and positive family history" are generally defined as "high-risk or high-risk people for cardia cancer" and are also the main targets for early screening of cardia cancer. At present, endoscopic examination, mucosal biopsy, histopathological examination, especially the census and follow-up of asymptomatic residents in high-incidence areas, are still the most effective means to detect patients with early cancer and precancerous lesions. However, as the scope of screening expands, these methods have exposed some problems, which restrict the further promotion of screening, such as the close relationship between the detection rate of endoscopic screening and the prevalence rate, the compliance of the subjects, the endoscopic screening technology and the level of histopathological diagnosis. The lack of effective biological indicators and methods for early warning and early diagnosis of high-risk groups in existing technologies is the main reason for the late diagnosis of patients with cardiac cancer in clinical practice and the high mortality rate.

[0004] The occurrence and development of cardia cancer is a multi-factor, multi-stage, slow development process. The research team led by Professor Wang Lidong of the First Affiliated Hospital of Zhengzhou University found in the laboratory research and follow-up that the digestive tract mucosa of patients with precancerous lesions of the cardia confirmed by endoscopic biopsy can be further transformed from atypical hyperplasia (precancerous lesions) to cancer; however, some people can stop at a certain stage and no longer develop further into malignancy, and even transform from precancerous lesions to normal epithelial tissue. This phenomenon suggests that precancerous lesions of the cardia have the characteristics of bidirectional development. In order to further reduce the mortality rate of cardia cancer and improve the prognosis of patients, it is still key to improve the early detection, early diagnosis and early treatment of cardia cancer, and it is particularly important to seek a new non-invasive diagnostic method that can be used for population screening.

[0005] For several years, our research team has been committed to studying the mechanism of the occurrence and development of cardiac cancer, and has also conducted a lot of research on the metabolites of cardiac cancer itself. Using the serum of patients with cardiac adenocarcinoma as the research object, two tumor metabolites (Xanthurenic Acid and 2-Hydroxy-2-methylbutyric acid) were screened out. The expression levels in the serum of patients with cardiac adenocarcinoma were significantly different from those in the normal control group, and they can be detected in the blood samples of the early stage of cardiac cancer and even precancerous lesions. Therefore, this application attempts to use Xanthurenic Acid and 2-Hydroxy-2-methylbutyric acid to achieve early screening and diagnosis of cardiac cancer, opening up new ways for the screening and diagnosis of cardiac cancer. Summary of the invention

[0006] In view of the problems and shortcomings of the prior art, one of the objects of the present invention is to provide a marker for screening for cardiac cancer. A second object of the present invention is to provide a detection reagent for the marker for screening for cardiac cancer for use in the preparation of a cardiac cancer screening product. A third object of the present invention is to provide a kit for screening for cardiac cancer.

[0007] To achieve the purpose of the invention, the technical solution adopted by the present invention is as follows:

[0008] A first aspect of the present invention provides a marker for screening for cardiac cancer, wherein the marker is at least one of Xanthurenic Acid and 2-Hydroxy-2-methylbutyric acid.

[0009] According to the above-mentioned marker, preferably, the marker is a serum marker.

[0010] The second aspect of the present invention provides a use of a detection reagent for the marker described in the first aspect in the preparation of a product for screening cardiac cancer.

[0011] According to the above application, preferably, the test sample of the product is serum.

[0012] According to the above application, preferably, the detection reagent is a reagent for detecting the marker in a sample by chromatography, mass spectrometry or chromatography-mass spectrometry.

[0013] According to the above application, preferably, the chromatography is gas chromatography, liquid chromatography or high performance liquid chromatography.

[0014] According to the above application, preferably, the chromatography-mass spectrometry is gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, high performance liquid chromatography-mass spectrometry, gas chromatography-tandem mass spectrometry, liquid chromatography-tandem mass spectrometry or high performance liquid chromatography-tandem mass spectrometry.

[0015] The third aspect of the present invention provides a kit for screening cardiac cancer, the kit comprising a detection reagent for detecting the marker described in the first aspect, wherein the marker is at least one of Xanthurenic Acid and 2-Hydroxy-2-methylbutyric acid.

[0016] According to the above-mentioned kit, preferably, the detection reagent is a reagent for detecting the marker in a sample by chromatography-mass spectrometry or chromatography-mass spectrometry.

[0017] According to the above-mentioned kit, preferably, the chromatography is gas chromatography, liquid chromatography or high performance liquid chromatography; the chromatography-mass spectrometry is gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, high performance liquid chromatography-mass spectrometry, gas chromatography-tandem mass spectrometry, liquid chromatography-tandem mass spectrometry or high performance liquid chromatography-tandem mass spectrometry.

[0018] According to the above-mentioned kit, preferably, the kit further contains a standard product of the marker described in the first aspect above.

[0019] According to the above-mentioned kit, preferably, the detection sample of the kit is serum.

[0020] Compared with the prior art, the present invention has the following positive and beneficial effects:

[0021] (1) The present invention first discovered through the metabolomics method that Xanthurenic Acid and 2-Hydroxy-2-methylbutyric acid can be used for early screening and detection of cardiac cancer. By detecting the expression levels of Xanthurenic Acid and 2-Hydroxy-2-methylbutyric acid in human serum, cardiac cancer, especially early cardiac cancer, can be effectively detected; it has been verified that when the present invention uses any one of Xanthurenic Acid and 2-Hydroxy-2-methylbutyric acid alone for cardiac cancer screening, the AUC value of the ROC curve is above 0.9; when Xanthurenic Acid and 2-Hydroxy-2-methylbutyric acid are used in combination, the AUC value of the ROC curve is closer to 1 than a single indicator, and the differentiation effect is good, and the diagnostic effect is good. Therefore, the marker for cardiac cancer screening of the present invention can be used for early screening of cardiac cancer.

[0022] (2) When the present invention uses the two markers of Xanthurenic Acid and 2-Hydroxy-2-methylbutyric acid as a combination for early cardiac cancer screening, the detection sensitivity is as high as 94.3% (i.e., the rate of early cardiac cancer patients correctly diagnosed with early cardiac cancer when these two markers are used for diagnosis is 94.3%), and the specificity is 97.0% (i.e., the rate of patients without cardiac cancer who are diagnosed as not suffering from cardiac cancer when these two markers are used for diagnosis is 97.0%). Therefore, the markers of the present invention have high sensitivity and specificity. It has greatly improved the detection rate of early cardiac cancer, and its detection rate is much higher than the existing clinical endoscopic screening detection rate of cardiac cancer (2%-3%). It can be used for large-scale screening of asymptomatic high-risk people in areas with a high incidence of cardiac cancer. It also provides an important detection method for long-term follow-up of asymptomatic high-risk people in areas with a high incidence of cardiac cancer, which is conducive to the early detection of asymptomatic high-risk people for cardiac cancer, thereby greatly reducing the mortality rate of patients with cardiac cancer and bringing great benefits to patients with cardiac cancer and their families.

[0023] (3) The marker used for screening of gastric cardia cancer in the present invention is a serum detection marker, which can avoid invasive diagnosis. The risk of gastric cardia cancer can be obtained at an early stage by taking serum for testing in a minimally invasive manner, thereby providing a basis for clinicians to conduct further in-depth examinations, and providing support for quickly and accurately understanding the patient's disease status and severity of the disease, and timely adopting more personalized prevention and treatment plans, thereby delaying and preventing disease progression.

[0024] (4) The test sample of the kit for screening cardia cancer of the present invention is serum, which requires a small amount of blood, causes little pain to the public, and has high acceptance; moreover, the operation is simple, the detection result takes a short time, and it has broad market prospects and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The ROC curve of using Xanthurenic Acid to diagnose and distinguish cardiac cancer patients from normal control group;

[0026] Figure 2 The ROC curve of using 2-Hydroxy-2-methylbutyric acid to diagnose and differentiate cardiac cancer patients from normal controls;

[0027] Figure 3 This is the ROC curve of the combination of Xanthurenic Acid and 2-Hydroxy-2-methylbutyric acid in distinguishing cardiac cancer patients from the normal control group. DETAILED DESCRIPTION

[0028] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings. It will be understood by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0029] The experimental methods described in the following examples, unless otherwise specified, are all conventional techniques in the art, or are based on the conditions recommended by the manufacturers; the reagents, materials and instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0030] Example 1: Screening of serum differential metabolic markers for cardiac cancer

[0031] 1. Experimental samples

[0032] Training set samples: 60 healthy subjects (normal control group) and 60 patients with cardia cancer (cardia cancer group) were collected from Henan Province and Anyang Cancer Hospital according to strict screening and exclusion criteria and matched by age and gender.

[0033] The inclusion criteria for healthy subjects were: no cardiovascular, respiratory, liver, kidney, gastrointestinal, endocrine, blood, psychiatric, or nervous system diseases or history of the above diseases, no acute or chronic diseases, no evidence of any tumor, no history of drug allergy, and clinical laboratory test results within the normal reference range at screening.

[0034] The inclusion criteria for patients with cardiac cancer were as follows: patients with cardiac cancer confirmed by endoscopy and histopathology who had not received radiotherapy or chemotherapy.

[0035] 2. Experimental methods

[0036] (1) Collection and storage of serum samples:

[0037] Collect peripheral blood from patients in the morning on an empty stomach and place it in a test tube without anticoagulant. Allow the blood to coagulate naturally for 30-60 minutes at room temperature. After the blood coagulates, centrifuge it at 2000 rpm for 10 minutes. Carefully draw the clear serum liquid from the upper layer into a sterile freeze-dried tube, mark it, and store it in a -80°C refrigerator for later use.

[0038] (2) Main reagents:

[0039] Methanol and acetonitrile (UPLC pure) were purchased from Merck, USA; chromatographic grade formic acid and ammonium acetate were purchased from ROE, USA; deionized water was prepared by the Mlli-Q ultrapure water system of Millipore, USA; standard substances including Xanthurenic Acid and 2-Hydroxy-2-methylbutyric acid were purchased from Sigma-Aldrich, USA.

[0040] (3) UPLC-Q / TOF-MS detection:

[0041] A) Testing instruments:

[0042] Agilent 1290 high performance liquid chromatograph and Agilent 6545 quadrupole-time of flight mass spectrometer.

[0043] B) Chromatographic conditions:

[0044] Chromatographic column: Waters BEH C8 (100mm×2.1mm ID, 1.7μm); positive ion mobile phase: acetonitrile (B)-water (A) solution (both containing 0.1% formic acid); negative ion mobile phase: methanol (B)-water (A) solution (both containing 10mmol / L ammonium acetate); flow rate: 0.4mL / min; column temperature: 50℃, injection volume: 1μL. Mobile phase ratio: 0-1min, 5% B; 1-4min, 5%-30% B; 4-9min, 30%-90% B; 9-10min, 90%-100% B; 10-12min, 100% B. The running time was 12 minutes, and the post-running time was 3 minutes.

[0045] C) Mass spectrometry conditions:

[0046] Fragmentation voltage: 100 V; nebulizing gas: 35 psig; capillary voltage: 3500 V; drying gas flow: 10 L / min; drying gas temperature: 300 °C, injection volume: 1 μL.

[0047] D) Sample processing:

[0048] Take 50 μL of serum sample, then add 150 μL of 3 times amount of methanol, vortex for 30 seconds to mix, and then put it into a high-speed centrifuge for centrifugation at 13000 rpm for 10 minutes. Take 75 μL of the supernatant after centrifugation and put it into two 1.5 ml centrifuge tubes, blow dry the supernatant with a nitrogen blower, and then re-dissolve it with 100 μL of methanol re-solution containing internal standard solution (L-2-chlorophenylalanine) or 100 μL of methanol re-solution containing internal standard solution (ketoprofen). The final concentrations of L-2-chlorophenylalanine and ketoprofen are 100 ng / mL and 1 μg / mL, respectively, and they are used as positive and negative ion mode detection samples, respectively. After re-dissolution, vortex for 30 seconds to mix, then place in a high-speed centrifuge for centrifugation at 13,000 rpm for 10 minutes, aspirate the supernatant after centrifugation, and then place the supernatant in a liquid chromatography-mass spectrometry vial for UPLC-Q / TOF-MS detection.

[0049] (4) Data processing method:

[0050] A) Data pre-processing:

[0051] Based on the data obtained by UPLC-Q / TOF-MS, XCMS program code was used under the R software platform for peak extraction, alignment and deconvolution analysis, and peak screening was performed according to the 80% principle to obtain a three-dimensional visualization matrix containing variables (retention time Rt, mass-to-charge ratio m / z), observations and peak intensities. This data matrix was imported into SIMCA-P software (version 13.0) for multivariate statistical analysis.

[0052] B) Multivariate statistical analysis:

[0053] In order to investigate the metabolic changes in the cardiac cancer group compared with the normal control group, unsupervised principal component analysis (PCA) was first performed on all variables to observe the clustering of data in each group and remove outliers. Finally, the orthogonal partial least squares discriminant analysis (PLS-DA) model was used for supervised data analysis to amplify the differences between the groups and obtain the most significant separation between the groups.

[0054] C) Mining and identification of differential metabolites between cardiac cancer group and normal control group based on UPLC-Q / TOF-MS detection:

[0055] The VIP values ​​of the cardiac cancer group and the normal control group under the PLS-DA model were combined with the P values ​​of the univariate statistical analysis. Variables with VIP>1.0 and P<0.05 were considered to have significant differences, and variables with significant differences were considered to be differential metabolic biomarkers.

[0056] The screened differential variables need to be attributed to the biomarkers they represent. Metabolite identification based on UPLC-Q / TOF-MS technology is mainly matched through metabolite spectral libraries: find the mass spectrum of the differential variable on the UPLC-Q / TOF-MS total ion flow chart, compare the precise molecular weight of the differential metabolite with the network database, such as HMDB (http: / / www.hmdb.ca), METLIN (http: / / metlin.scripps.edu) and KEGG (http: / / www.kegg.jp), preliminarily identify the structure of the above differential metabolites, and finally determine the structure of the differential metabolites by purchasing standard products, using the molecular weight, chromatographic retention time and corresponding multi-stage MS fragmentation spectrum of the standard products, and prepare a series of standard solutions with known concentrations, and further determine the content of the differential metabolites through the standard curve.

[0057] 3. Experimental results

[0058] According to the above experimental method, two differential metabolites were finally identified that distinguished the cardiac cancer group from the normal control group, as shown in Table 1. Moreover, compared with the normal control group, the levels of Xanthurenic Acid and 2-Hydroxy-2-methylbutyric acid in the serum of patients with cardiac cancer were significantly increased.

[0059] Table 1 Statistical data of differential metabolites between cardiac cancer group and normal control group based on UPLC-Q / TOF-MS

[0060] serial number Differential metabolites VIP P-value Multiple 1 Xanthurenic Acid 1.9 0.041 7.20 2 2-Hydroxy-2-methylbutyric acid 2.2 0.035 5.43

[0061] Example 2: Evaluation of the ability of differential metabolite diagnosis to distinguish cardiac cancer patients from healthy subjects

[0062] (1) The ability of a single differential metabolite to diagnose and distinguish patients with gastric cardia cancer from normal subjects:

[0063] Based on the UPLC-Q / TOF-MS detection in Example 1, the serum samples of the cardiac cancer group (60 cardiac cancer patients) and the normal control group (60 normal people) were Xanthurenic Acid,

[0064] The content data of 2-Hydroxy-2-methylbutyric acid were analyzed, and the receiver operating characteristic curve (ROC curve) was used to evaluate the ability of each differential metabolite to diagnose and distinguish cardiac cancer patients from normal subjects.

[0065] The ROC curves of xanthurenic acid and 2-Hydroxy-2-methylbutyric acid for distinguishing cardiac cancer patients from normal subjects are shown below. Figure 1 , Figure 2 According to the ROC curve, the area under the curve (AUC), sensitivity and specificity of the ROC curve of each differential metabolite were statistically analyzed, and the results are shown in Table 2.

[0066] Table 2 AUC of two differential metabolites for distinguishing cardiac cancer patients from normal subjects

[0067] serial number Differential metabolites AUC Sensitivity Specificity 1 Xanthurenic Acid 0.923 87.5％ 89.2％ 2 2-Hydroxy-2-methylbutyric acid 0.947 87.5％ 90.8％

[0068] The area under the ROC curve (AUC) has been widely recognized as an inherent accuracy indicator for evaluating the authenticity of diagnostic tests. When AUC is 0.5, it means that there is no diagnostic significance; when AUC is between 0.5 and 0.7, it means that the diagnostic accuracy is low; when AUC is between 0.7 and 0.9, it means that the diagnostic accuracy is moderate; when AUC>0.9, it means that the diagnosis has a high accuracy. Figure 2 As shown in Table 1, Xanthurenic Acid,

[0069] The AUC of the ROC curve of Xanthurenic Acid, 2-Hydroxy-2-methylbutyric acid alone for distinguishing patients with cardiac cancer from normal subjects was above 0.9, indicating that

[0070] 2-Hydroxy-2-methylbutyric acid can independently diagnose and differentiate patients with gastric cardia cancer from normal persons with good accuracy.

[0071] The Youden index (Youden index = sensitivity + specificity - 1) was further calculated based on the coordinates of the ROC curve. The relative content of the metabolites corresponding to the maximum Youden index was the optimal cutoff value for diagnosing and distinguishing patients with cardia cancer from normal people, as shown in Table 3.

[0072] Table 3 Youden index and cutoff value of two differential metabolites for distinguishing cardiac cancer patients from normal subjects

[0073] serial number Differential metabolites Youden Index Optimal cutoff value 1 Xanthurenic Acid 0.767 0.504 2 2-Hydroxy-2-methylbutyric acid 0.783 0.461

[0074] (2) The ability of the two differential metabolite combinations to diagnose and distinguish cardiac cancer patients from normal subjects:

[0075] The relative contents of Xanthurenic Acid and 2-Hydroxy-2-methylbutyric acid in the serum samples of the cardiac cancer group (60 cardiac cancer patients) and the normal control group (60 normal people) detected by UPLC-Q / TOF-MS in Example 1 were used as independent variables (assuming X1=relative content of Xanthurenic Acid, X2=relative content of 2-Hydroxy-2-methylbutyric acid), and the groups (cardiac cancer group and normal control group) were used as dependent variables. Binary logistic regression was performed on the relative contents of Xanthurenic Acid and 2-Hydroxy-2-methylbutyric acid in the serum samples of the cardiac cancer group and the normal control group to obtain a binary logistic regression equation: logit[p]=11.35-5.353X1-75.46X2; the relative contents of Xanthurenic Acid and 2-Hydroxy-2-methylbutyric acid in each serum sample were then calculated. Substituting the relative content of acid into the binary logistic regression equation, the regression value logit[p] of each serum sample can be obtained. The possible regression value logit[p] is used as the diagnostic point to calculate the sensitivity and specificity (specificity is also called specificity). Then, the ROC curve is drawn based on the calculated sensitivity and specificity. The ROC curve is shown in Figure 2 shown.

[0076] From the ROC curve, it can be seen that the area under the ROC curve AUC of MG (22:6) and urocanic acid combined to diagnose and distinguish patients with gastric cardia cancer from normal people is 0.983, which has high accuracy. The Youden index (Youden index = sensitivity + specificity - 1) was further calculated based on the coordinates of the ROC curve. The logit [p] value corresponding to the maximum Youden index was the optimal cutoff value for diagnosing and distinguishing patients with gastric cardia cancer from normal people, and the optimal cutoff value was 0.468.

[0077] The ROC curve AUC values, sensitivity, specificity, Youden index and optimal cutoff value for the above single and two differential metabolites combined to distinguish cardiac cancer patients from normal subjects were statistically analyzed, as shown in Table 4.

[0078] Table 4 AUC values ​​of different differential metabolite combinations for diagnosing and distinguishing cardiac cancer patients from normal subjects

[0079]

[0080] As shown in Table 4, compared with a single differential metabolite, when the two differential metabolites of Xanthurenic Acid and 2-Hydroxy-2-methylbutyric acid were used to diagnose and distinguish between patients with gastric cardia cancer and normal people, the area under the ROC curve AUC reached 0.983, which was significantly higher than the diagnosis of a single differential metabolite, and the diagnostic sensitivity (94.3%) and specificity (97.0%) of gastric cardia cancer also reached the highest, indicating that the combination of Xanthurenic Acid and 2-Hydroxy-2-methylbutyricacid had the best diagnostic effect on gastric cardia cancer. In addition, the Youden index in statistics refers to the sum of sensitivity and specificity minus 1, and its value range is 0 to 1. The closer the Youden index is to 1, the greater its diagnostic value, indicating that the application value of this method is higher. When Xanthurenic Acid and 2-Hydroxy-2-methylbutyric acid are combined, their Youden index is significantly higher than that of a single differential metabolite, indicating that the method of combining the two differential metabolites of Xanthurenic Acid and 2-Hydroxy-2-methylbutyric acid for diagnosing cardiac cancer has a good diagnostic value.

[0081] Example 3: Application of two differential metabolites in cardiac cancer screening

[0082] 1. Collection of serum samples

[0083] A total of 580 healthy subjects (normal control group) and 437 patients with cardiac cancer (cardiac cancer group) were collected from Henan Province and Anyang Cancer Hospital according to strict screening and exclusion criteria.

[0084] The inclusion criteria for healthy subjects were: no cardiovascular, respiratory, liver, kidney, gastrointestinal, endocrine, blood, psychiatric, or nervous system diseases or history of the above diseases, no acute or chronic diseases, no evidence of any tumor, no history of drug allergy, and clinical laboratory test results within the normal reference range at screening.

[0085] The inclusion criteria for patients with cardiac cancer were as follows: patients with cardiac cancer confirmed by endoscopy and histopathology who had not received radiotherapy or chemotherapy.

[0086] 2. Experimental methods

[0087] (1) Collection and storage of serum samples:

[0088] Collect peripheral blood from patients in the morning on an empty stomach and place it in a test tube without anticoagulant. Allow the blood to coagulate naturally for 30-60 minutes at room temperature. After the blood coagulates, centrifuge it at 2000 rpm for 10 minutes. Carefully draw the clear serum liquid from the upper layer into a sterile freeze-dried tube, mark it, and store it in a -80°C refrigerator for later use.

[0089] 3. Experimental and analytical methods

[0090] According to the UPLC-Q / TOF-MS experimental method described in Example 1, the sera of 580 healthy subjects (normal control group) and 437 cardiac cancer patients (cardiac cancer group) were qualitatively and quantitatively determined for two metabolites (Xanthurenic Acid and 2-Hydroxy-2-methylbutyric acid).

[0091] When Xanthurenic Acid or 2-Hydroxy-2-methylbutyric acid is used alone for the diagnosis of cardiac cancer, the negative and positive of the sample are judged according to the content of Xanthurenic Acid or 2-Hydroxy-2-methylbutyric acid in the serum sample and the optimal cutoff value of the corresponding differential metabolite calculated in Example 2. If the content of the differential metabolite in the serum sample is higher than the optimal cutoff value, the patient is judged to be a cardiac cancer patient, otherwise, the patient is judged to be a normal person.

[0092] When the two metabolic markers of Xanthurenic Acid and 2-Hydroxy-2-methylbutyric acid are used in combination for the diagnosis of cardiac cancer, the contents of Xanthurenic Acid and 2-Hydroxy-2-methylbutyric acid in the serum sample are substituted into the Logistic regression equation obtained in Example 2. The negative and positive of the sample are judged according to the calculated logit [p] value and the optimal cutoff value of the combination of Xanthurenic Acid and 2-Hydroxy-2-methylbutyric acid calculated in Example 2 for distinguishing cardiac cancer patients from normal persons. If the logit [p] value is higher than the optimal cutoff value of 0.468, it is judged as cardiac cancer. Otherwise, it is judged as a normal person.

[0093] 3. Test results

[0094] Table 5 Results of the combination of two metabolic markers in diagnosing cardiac cancer

[0095]

[0096] The test results are shown in Table 5. When Xanthurenic Acid or 2-Hydroxy-2-methylbutyricacid is used alone for the diagnosis of cardiac cancer, the positive predictive value can reach more than 88%, and the negative predictive value can reach more than 89%; moreover, when Xanthurenic Acid and 2-Hydroxy-2-methylbutyric acid are used together for the diagnosis of cardiac cancer, the positive predictive value and negative predictive value are both the highest, 92.93% and 95.42%, respectively. It can be seen that the combined diagnosis of cardiac cancer with Xanthurenic Acid and 2-Hydroxy-2-methylbutyric acid is the most effective.

[0097] Example 4: Preparation of a Cardiac Cancer Screening Kit Based on the Metabolic Markers of the Present Invention

[0098] Based on the two metabolic markers related to cardiac cancer obtained by screening of the present invention, a cardiac cancer screening kit is designed, and the kit includes the following components:

[0099] Marker standard: at least one of Xanthurenic Acid and 2-Hydroxy-2-methylbutyric acid. The kit may contain one or two metabolic marker standard products, which may be combined as required. When two metabolic marker standard products are included, the two marker standard products may be packaged separately, or the two marker standard products may be mixed to form a mixture and packaged.

[0100] Internal standard: The internal standard is L-2-chlorophenylalanine.

[0101] Furthermore, the kit also contains a serum metabolite extraction solvent, methanol, and a mobile phase reagent used in the UPLC-Q / TOF-MS detection process (the mobile phase reagent is the same as the mobile phase reagent used in the chromatographic detection process of Example 1).

[0102] The use process of the kit is as follows: collect the serum of the subjects and freeze it in a -80℃ refrigerator. Before the experiment, thaw the serum sample in a 4℃ refrigerator, take 50μL of the serum sample, then add 150μL of 3 times the amount of methanol, vortex for 30s to mix, and then put it into a high-speed centrifuge for centrifugation at 13000rpm for 10min. Take 75μL of the supernatant after centrifugation and put it into two 1.5ml centrifuge tubes, blow dry the supernatant with a nitrogen blower, and then re-dissolve it with 100μL of methanol re-solution containing internal standard solution (L-2-chlorophenylalanine), vortex for 30s to mix, and then put it into a high-speed centrifuge for centrifugation at 13000rpm for 10min. Take the supernatant after centrifugation and put it into a liquid mass spectrometer vial for UPLC-Q / TOF-MS detection. The UPLC-Q / TOF-MS detection is performed by analyzing the treated serum samples according to the UPLC-Q / TOF-MS instrument setting method of Example 1, and performing quantitative and qualitative analysis of the markers according to the data processing method of Example 1.

[0103] When using this cardia cancer detection kit, it is recommended to simultaneously detect two markers, Xanthurenic Acid and 2-Hydroxy-2-methylbutyric acid, to further improve the detection efficiency.

[0104] In summary, the present invention effectively overcomes the deficiencies in the prior art and has a high industrial utilization value. The above embodiments serve to illustrate the substantive content of the present invention, but do not limit the protection scope of the present invention. Those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and protection scope of the technical solution of the present invention.

Claims

1. Use of a 2-Hydroxy-2-methylbutyric detection reagent in the preparation of a cardiac cancer screening product, wherein the detection sample of the product is serum.

2. The use according to claim 1, characterized in that: The detection reagent is a reagent for detecting 2-Hydroxy-2-methylbutyric in a sample by chromatography-mass spectrometry.

3. The use according to claim 2, characterized in that: The chromatography-mass spectrometry method is liquid chromatography-mass spectrometry.

4. The use according to any one of claims 1 to 3, characterized in that: The product is a test kit.

5. The use according to claim 4, characterized in that: The kit also contains a standard of 2-Hydroxy-2-methylbutyric acid.

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