CN115825290A - Application of 2-Hydroxy-2-methylbutaric acid in cardiac cancer screening - Google Patents

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses a marker for cardiac cancer screening. The marker for screening cardiac cancer provided by the invention is 2-Hydroxy-2-methylbutaric acid, and a detection reagent of the marker can be used for preparing a cardiac cancer screening product. By detecting the expression level of 2-Hydroxy-2-methylbutaric acid in human serum, cardia cancer can be effectively detected, and the method can be used for large-scale screening of asymptomatic people in a cardia cancer high incidence area and is beneficial to screening and early discovery of the asymptomatic high risk people.

Description

Application of 2-Hydroxy-2-methylbutaric acid in cardiac cancer screening

The application is a divisional application of an invention patent with the application number of CN 202110717684.7, the application date of 2021-06-28 and the name of 'a marker and a detection kit for cardiac cancer screening'.

Technical Field

The invention belongs to the technical field of medical biology, and particularly discloses a marker for cardiac cancer screening and application thereof.

Background

With the development of society and the change of environment, cardiac cancer has become a malignant tumor that seriously threatens the health and safety of human life. Cardia cancer refers to cancer occurring in the anatomical region of the cardia, which is generally considered to be located within 2cm below the junction of the esophagus and the stomach in China, and is also called cardia adenocarcinoma. The researches show that the high incidence of the cardia cancer exists in the high incidence area of the esophageal cancer. This phenomenon suggests that in the local living environment, strong carcinogenic factors may exist, which act on the mucosa of the upper digestive tract for a long time, and lead to high incidence of both esophageal cancer and cardiac cancer through some similar mechanisms. Traditionally, "asymptomatic people with high incidence area, over 40 years old, male, smoking, drinking and positive family history" is generally defined as "people with high risk or high risk of cardiac cancer", and is also a main object for early screening of cardiac cancer. At present, endoscopy, mucosal biopsy and histopathology examination, particularly the general survey and follow-up of asymptomatic residents in high incidence areas, still remain the most effective means for finding patients with early cancer and precancerous lesions. However, with the expansion of the screening range, the methods expose some problems, which restrict the further popularization of the screening, such as the detection rate and the present disease rate of the endoscopic screening, the compliance of the examined person, the endoscopic screening technology and the histopathological diagnosis level have close relations. The lack of effective biological indexes and methods for early warning and early diagnosis of high risk groups in the prior art is a main reason for causing the late time of clinic cardiac cancer patients and high mortality.

The occurrence and development of cardia cancer are a multi-factor, multi-stage and slow development process, and a research team in the leaders of the professor Wanglidong of the first subsidiary hospital of Zhengzhou university finds that the cardia cancer is determined as a patient with precancerous lesion through endoscopic biopsy, and the digestive tract mucosa of the population can be further converted into cancer from atypical hyperplasia (precancerous lesion); however, some people can stop when they progress to a certain stage, and do not progress further to malignancy, and even can change from precancerous lesion to normal epithelial tissue, which suggests that cardiac precancerous lesion has the characteristic of bidirectional development. In order to further reduce the mortality rate of cardiac cancer, improve the prognosis of patients and improve the early detection, early diagnosis and early treatment of cardiac cancer, it is important to find a new non-invasive diagnosis method for population screening.

For years, the research team has been devoted to the research on the mechanism of occurrence and development of cardiac cancer, and a great deal of research on the metabolites of cardiac cancer itself has been carried out. By taking the serum of a patient suffering from cardia adenocarcinoma as a research object, 2 tumor metabolites (Xanthurenic Acid and 2-Hydroxy-2-methylbutastic Acid) are screened out, the expression level of the serum of the patient suffering from cardia adenocarcinoma is obviously different from that of a normal control group, and the serum can be detected in the early stage of cardia cancer and even in a blood sample of precancerous lesion, so that the method tries to realize the early screening and diagnosis of cardia cancer by using the Xanthurenic Acid and the 2-Hydroxy-2-methylbutastic Acid, and develops a new way for the screening and diagnosis of cardia cancer.

Disclosure of Invention

In view of the problems and disadvantages of the prior art, an object of the present invention is to provide a marker for cardiac cancer screening, a second object of the present invention is to provide a use of a detection reagent for the marker for cardiac cancer screening in the preparation of a cardiac cancer screening product, and a third object of the present invention is to provide a kit for cardiac cancer screening.

In order to realize the purpose of the invention, the technical scheme adopted by the invention is as follows:

the invention provides a marker for cardiac cancer screening, wherein the marker is at least one of Xanthus Acid and 2-Hydroxy-2-methylbutanic Acid.

Preferably, the marker is a serum marker according to the above.

In a second aspect, the present invention provides a use of a detection reagent for the marker of the first aspect in the preparation of a product for screening cardiac cancer.

According to the above-mentioned application, preferably, the test sample of the product is serum.

According to the above-mentioned use, preferably, the detection reagent is a reagent for detecting the marker in the sample by chromatography, mass spectrometry or a combination of chromatography and mass spectrometry.

According to the above-mentioned use, preferably, the chromatography is gas chromatography, liquid chromatography or high performance liquid chromatography.

According to the above application, preferably, the chromatography-mass spectrometry combination is a gas chromatography-mass spectrometry combination, a liquid chromatography-mass spectrometry combination, a high performance liquid chromatography-mass spectrometry combination, a gas chromatography-tandem mass spectrometry combination, a liquid chromatography-tandem mass spectrometry combination or a high performance liquid chromatography-tandem mass spectrometry combination.

In a third aspect, the present invention provides a kit for cardiac cancer screening, said kit comprising a detection reagent for detecting the marker of the first aspect. The marker is at least one of Xanthus Acid and 2-Hydroxy-2-methylbutanic Acid.

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

Preferably, the chromatography is gas chromatography, liquid chromatography or high performance liquid chromatography according to the above kit; the chromatography-mass spectrometry combined method is a gas chromatography-mass spectrometry combined method, a liquid chromatography-mass spectrometry combined method, a high performance liquid chromatography-mass spectrometry combined method, a gas chromatography-tandem mass spectrometry combined method, a liquid chromatography-tandem mass spectrometry combined method or a high performance liquid chromatography-tandem mass spectrometry combined method.

Preferably, the kit further comprises a standard for the marker of the first aspect.

According to the kit, preferably, the detection sample of the kit is serum.

Compared with the prior art, the invention has the following positive beneficial effects:

(1) According to the invention, the two substances of Xanthus Acid and 2-Hydroxy-2-methylbutanic Acid are found to be capable of being used for early screening and detecting cardiac cancer for the first time by a metabonomics method, and the cardiac cancer, especially early cardiac cancer, can be effectively detected by detecting the expression levels of Xanthus Acid and 2-Hydroxy-2-methylbutanic Acid in human serum; according to verification, when any marker of Xanthurenic Acid and 2-Hydroxy-2-methylbutanic Acid is independently adopted for cardiac cancer screening, the AUC value of the ROC curve is above 0.9; when two markers of Xanthus Acid and 2-Hydroxy-2-methylbutanic Acid are used together, the AUC value of the ROC curve is closer to 1 than that of a single marker, the distinguishing effect is good, and the diagnosis effect is good. Therefore, the marker for cardiac cancer screening can be used for early-stage cardiac cancer screening.

(2) The invention uses two markers of Xanthus panicus Acid and 2-Hydroxy-2-methylbutaric Acid as a combination for screening and detecting early-stage cardia cancer, the detection sensitivity is as high as 94.3% (namely the rate of correctly diagnosing the early-stage cardia cancer in an early-stage cardia cancer patient by using the two markers is 94.3%), the specificity reaches 97.0% (namely the rate of determining the patient without cardia cancer in a non-cardia cancer patient by using the 2 markers for diagnosis is 97.0%), therefore, the marker has higher sensitivity and specificity, the detection rate of the early-stage cardia cancer is greatly improved, the detection rate of the cardia cancer is far higher than that of the conventional clinical endoscopic screening, the marker can be used for large-scale screening of people at high risk of cardia cancer, and also provides an important means for realizing long-term tracking of the high risk of cardia cancer, thereby being beneficial to the family high-risk cancer patients and greatly reducing the early-stage cardia cancer death rate.

(3) The marker for screening cardiac cancer is a serum detection marker, so that invasive diagnosis can be avoided, and the cardiac cancer risk can be obtained at an early stage by taking serum for detection in a minimally invasive way, so that a basis is provided for further in-depth examination by a clinician, support is provided for rapidly and accurately mastering the disease state and the severity of the condition of a patient, a more personalized prevention and treatment scheme is adopted in time, and the disease progress is delayed and prevented.

(4) The detection sample of the kit for screening cardiac cancer is serum, so that the blood demand is low, the pain of the masses is small, and the acceptability is high; moreover, the method is simple to operate, short in detection result time and wide in market prospect and social benefit.

Drawings

FIG. 1 is a ROC plot of a patient group with cardiac cancer and a normal control group differentiated by Xanthurenic Acid diagnosis;

FIG. 2 is a ROC graph for distinguishing a cardiac cancer patient group and a normal control group by using a 2-Hydroxy-2-methylbutatic acid diagnosis;

FIG. 3 is a ROC graph of combined diagnosis of Xanthus Acid and 2-Hydroxy-2-methylbutanic Acid for differentiating cardiac cancer patient groups from normal control groups.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the following detailed description and accompanying drawings. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention.

The experimental procedures described in the following examples, unless otherwise specified, are conventional in the art or according to the conditions recommended by the manufacturers; the reagents, materials and instruments used are not indicated by manufacturers, and are all conventional products commercially available.

Example 1: screening of cardia cancer serum differential metabolic markers

1. Experimental sample

Training set samples: tumor hospitals in the regions of Henan and Anyang were collected and 60 healthy subjects (normal control group) and 60 patients with cardiac cancer (cardiac cancer group) were age-and sex-matched according to strict screening and exclusion criteria.

The inclusion criteria for healthy subjects were: there is no cardiovascular, respiratory, liver, kidney, gastrointestinal, endocrine, blood, mental, or nervous system disease and history of the above diseases, no acute or chronic disease, no evidence of any tumor-related disease, no history of drug allergy, and clinical laboratory test results in the screening are within the normal reference range.

The group entry criteria for patients with cardiac cancer were: patients with cardia cancer who are determined by endoscopy and confirmed by histopathology do not receive radiotherapy or chemotherapy.

2. Experimental methods

(1) Collecting and storing serum samples:

collecting fasting peripheral blood of a patient in the early morning, placing the fasting peripheral blood in a test tube without anticoagulant, naturally coagulating for 30-60min at room temperature, after blood coagulation, centrifuging for 10min at 2000rpm, carefully sucking supernatant clear serum liquid into a sterile freeze-drying tube, marking, and storing in a refrigerator at-80 ℃ for later use.

(2) The main reagents are as follows:

methanol and acetonitrile (UPLC pure) from merk, usa, and chromatographic grade formic acid and ammonium acetate from ROE, usa; deionized water was prepared from the Milli-Q ultrapure water system from Millipore, inc., USA; standards include Xanthurenic Acid and 2-Hydroxy-2-methylbutanic Acid available from Sigma-Aldrich, USA.

(3) UPLC-Q/TOF-MS detection:

a) And (3) detecting an instrument:

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

B) Chromatographic conditions are as follows:

a chromatographic column: waters BEH C8 (100 mm × 2.1mm i.d.,1.7 μm); acetonitrile (B) -water (a) solutions of the cationic mobile phase (both containing 0.1% formic acid); negative ion mobile phase methanol (B) -water (A) solutions (both containing 10mmol/L ammonium acetate); flow rate: 0.4mL/min; column temperature: the sample size was 1. Mu.L at 50 ℃. Mobile phase ratio: 0-1min,5% by weight B;1-4min,5% -30% B;4-9min,30% -90% B;9-10min,90% -100% of B;10-12min,100% B. The run time was 12 minutes and the post run time was 3 minutes.

C) Mass spectrum conditions:

fragment voltage: 100V; atomizing gas: 35psig; capillary voltage: 3500V; flow rate of drying gas: 10L/min; drying gas temperature: the sample size was 1. Mu.L at 300 ℃.

D) Sample treatment:

taking 50 mu L of serum sample, adding 150 mu L of methanol with the volume being 3 times that of the serum sample, vortexing for 30s, uniformly mixing, putting into a high-speed centrifuge for centrifugation, and centrifuging for 10min at 13000 rpm. And sucking 75 mu L of centrifuged supernatant, respectively putting the supernatant into 2 centrifuge tubes with the volume of 1.5mL, drying the supernatant by using a nitrogen blower, re-dissolving the supernatant by respectively using 100 mu L of methanol complex solution containing an internal standard solution (L-2-chlorophenylalanine) or 100 mu L of methanol complex solution containing an internal standard solution (ketoprofen) after drying, wherein the final concentrations of the L-2-chlorophenylalanine and the ketoprofen are respectively 100ng/mL and 1 mu g/mL, and the final concentrations are respectively used as positive and negative ion mode detection samples. And (3) after redissolving, uniformly mixing by vortexing for 30s, putting the mixture into a high-speed centrifuge for centrifugation at 13000rpm for 10min, sucking the centrifuged supernatant, and then putting the supernatant into a liquid vial for UPLC-Q/TOF-MS detection.

(4) The data processing method comprises the following steps:

a) Preprocessing data:

based on data obtained by UPLC-Q/TOF-MS, under an R software platform, XCMS program codes are adopted for extracting peaks, aligning and deconvoluting analysis, and the peaks are screened according to the principle of 80% to obtain a three-dimensional visual matrix containing variables (retention time Rt, mass-to-charge ratio m/z), observation and peak intensity, and the data matrix is imported into SIMCA-P software (version 13.0) for multivariate statistical analysis.

B) Multivariate statistical analysis:

in order to examine the metabolic change of the cardiac cancer group compared with the normal control group, unsupervised Principal Component Analysis (PCA) is firstly adopted for all variables, the clustering condition of each group of data is observed, the outlier is removed, finally, supervised data analysis is carried out by adopting a least square method of orthogonality discriminant analysis (PLS-DA) model, and the difference among groups is amplified so as to obtain the most obvious separation among the groups.

C) And (3) detecting excavation and identification of differential metabolites between the cardia cancer group and the normal control group by using UPLC-Q/TOF-MS:

the VIP values under the PLS-DA model of the cardiac cancer group and the normal control group are combined with the P value analyzed by the single factor statistics, the variable with VIP >1.0 and P <0.05 is considered to have significant difference, and the variable with significant difference is considered to be a differential metabolism biomarker.

The selected differential variable needs to be assigned to the biomarker it represents. Metabolite identification based on UPLC-Q/TOF-MS technology is mainly by matching via a library of metabolite profiles: finding a mass spectrogram of a difference variable on a UPLC-Q/TOF-MS total ion flow graph, comparing the accurate molecular weight of a difference metabolite with a network database, such as HMDB (http:// www.hmdb.ca), METLIN (http:// METLIN. Script.edu) and KEGG (http:// www.kegg.jp), preliminarily identifying the structure of the difference metabolite, finally, determining the structure of the difference metabolite by purchasing a standard, comparing the molecular weight of the standard, chromatographic retention time and a corresponding multi-stage MS cracking spectrum, preparing a series of standard solutions with known concentrations, and further determining the content of the difference metabolite through a standard curve.

3. Results of the experiment

According to the above experimental method, 2 differential metabolites were finally identified to distinguish the cardiac cancer group from the normal control group, as shown in table 1. Moreover, compared with a normal control group, the content of Xanthus Acid and 2-Hydroxy-2-methylbutanic Acid in the serum of the patient of the cardiac cancer group is obviously up-regulated.

TABLE 1 statistical data of the metabolite difference between the detection of cardiac cancer group and the normal control group based on UPLC-Q/TOF-MS

Numbering	Differential metabolites	VIP	P value	Multiple value
					1	Xanthurenic Acid	1.9	0.041	7.20
2	2-Hydroxy-2-methylbutyric acid	2.2	0.035	5.43

Example 2: assessment of the ability of differential metabolite diagnosis to distinguish between patients with cardiac cancer and healthy persons

(1) The ability of a single differential metabolite diagnosis to distinguish between patients with cardiac cancer and normal:

xanthurenic Acid, A.C. in serum samples of the cardia cancer group (60 patients with cardia cancer) and the normal control group (60 normal persons) based on the UPLC-Q/TOF-MS detection in example 1,

The content data of 2-Hydroxy-2-methylbutaric acid is analyzed, and a receiver operating curve (ROC curve) is adopted to evaluate the capability of each differential metabolite to distinguish cardiac cancer patients from normal persons by means of independent diagnosis.

The ROC curves of Xanthus Acid and 2-Hydroxy-2-methylbutaric Acid for distinguishing cardiac cancer patients from normal persons when they are diagnosed alone are shown in FIG. 1 and FIG. 2, respectively. According to the ROC curve, the area under the curve, AUC, sensitivity and specificity of the ROC curve of each differential metabolite are calculated, and the results are shown in Table 2.

TABLE 2 AUC for differential metabolite diagnosis alone to differentiate patients with cardiac cancer from normal

Numbering	Differential metabolites	AUC	Sensitivity of the probe	Degree of specificity
					1	Xanthurenic Acid	0.923	87.5％	89.2％
2	2-Hydroxy-2-methylbutyric acid	0.947	87.5％	90.8％

The area AUC under the ROC curve is generally accepted as the inherent accuracy index of the authenticity evaluation of the diagnostic test, and when the AUC is 0.5, the diagnostic significance is not realized; when the AUC is 0.5-0.7, the diagnosis accuracy is low; when the AUC is 0.7-0.9, the diagnosis accuracy is moderate; AUC > 0.9, indicating higher accuracy of diagnosis. As can be seen from FIG. 2 and Table 1, xanthurenic Acid,

The AUC of the ROC curve of the 2-Hydroxy-2-methylbutaric Acid which is used for distinguishing cardiac cancer patients from normal people independently can reach more than 0.9, which shows that Xanthurenic Acid,

The 2-Hydroxy-2-methylbutaric acid can be used for diagnosing and distinguishing cardiac cancer patients and normal people independently and has better accuracy.

Further, a jotan index (jotan index = sensitivity + specificity-1) was calculated from the coordinates of the ROC curve, and the relative metabolite content at the maximum jotan index was the optimal cutoff value for diagnosing and distinguishing cardiac cancer patients from normal persons, as shown in table 3.

TABLE 3 Jordan index and cutoff for the independent diagnosis of two different metabolites to differentiate cardiac cancer patients from normal

Numbering	Differential metabolites	Joden index	Optimum cut-off value
				1	Xanthurenic Acid	0.767	0.504
2	2-Hydroxy-2-methylbutyric acid	0.783	0.461

(2) Two differential metabolite combination diagnostics differentiate cardiac cancer patients from normal persons:

relative amounts of Xanthinic Acid and 2-Hydroxy-2-methylbutanic Acid in serum samples of the cardia cancer group (60 patients with cardia cancer) and the normal control group (60 normal persons) detected by UPLC-Q/TOF-MS in example 1 were used as independent variables (X is set) ₁ Relative content of = Xanthurenic Acid, X ₂ = 2-Hydroxy-2-methylbutaric Acid relative content), taking the group (cardiac cancer group and normal control group) as dependent variable, performing binary logistic regression on the relative content of Xanthus Acid and 2-Hydroxy-2-methylbutaric Acid in the cardiac cancer group and normal control group serum samples to obtain a binary logistic regression equation: logit [ p ]]＝11.35-5.353X ₁ -75.46X ₂ (ii) a Then each serum is sampledSubstituting the relative contents of Xanthus Acid and 2-Hydroxy-2-methylbutanic Acid into the binary logistic regression equation to obtain the regression value logic [ p ] of each serum sample]With possible regression values logit [ p ]]As a diagnosis point, the sensitivity and specificity (specificity is also called specificity) were calculated, and then a ROC curve was drawn based on the calculated sensitivity and specificity, and the ROC curve is shown in FIG. 2.

From the ROC curve, it is found that the combination diagnosis of MG (22) and urocanic acid has a high accuracy in distinguishing the area AUC under the ROC curve between the cardiac cancer patient and the normal person of 0.983. Further, a john's index (john's index = sensitivity + specificity-1) is calculated from the coordinates of the ROC curve, and the optimal cutoff value is 0.468, where the logit [ p ] value corresponding to the maximum john's index is the optimal cutoff value for diagnosing and distinguishing cardiac cancer patients from normal persons.

The combination diagnosis of single or two different metabolites can be used for distinguishing the AUC value, sensitivity, specificity, john's index and optimum cut-off value of ROC curve of cardiac cancer patient and normal person, and is specifically shown in Table 4

TABLE 4 AUC values for different differential metabolite combinations for diagnostic differentiation of patients with cardiac cancer from normal

As can be seen from Table 4, compared with a single differential metabolite, when a cardia cancer patient and a normal patient are diagnosed and distinguished by combining the Xanthhurinic Acid and the 2-Hydroxy-2-methylbutyric Acid, the AUC (area under the ROC curve) reaches 0.983 which is obviously higher than that of the diagnosis of the single differential metabolite, and the diagnosis sensitivity (94.3%) and specificity (97.0%) of the cardia cancer also reach the highest, which indicates that the diagnosis effect of the combination of the Xanthhurinic Acid and the 2-Hydroxy-2-methylbutyric Acid on the cardia cancer is the best. In addition, the jotan index is a value obtained by subtracting 1 from the sum of sensitivity and specificity in statistics, and is in a range of 0 to 1, and the closer the jotan index is to 1, the higher the diagnostic value is, indicating that the application value of the method is higher. When Xanthus Acid and 2-Hydroxy-2-methylbutaric Acid are combined, the Jordan index of the Xanthus Acid and 2-Hydroxy-2-methylbutaric Acid is obviously higher than that of a single differential metabolite, and the method for diagnosing cardiac cancer by combining the Xanthus Acid and the 2-Hydroxy-2-methylbutaric Acid has better diagnostic value.

Example 3: application of two different metabolites in cardiac cancer screening

1. Collection of serum samples

580 healthy subjects (normal control group) and 437 cardia cancer patients (cardia cancer group) were collected from tumor hospitals in Henan and Anyang city according to strict screening and exclusion criteria.

The inclusion criteria for healthy subjects were: there is no cardiovascular, respiratory, liver, kidney, gastrointestinal, endocrine, blood, mental, or nervous system disease and history of the above diseases, no acute or chronic disease, no evidence of any tumor-related disease, no history of drug allergy, and clinical laboratory test results in the screening are within the normal reference range.

The group entry criteria for patients with cardiac cancer were: patients with cardia cancer who are determined by endoscopy and confirmed by histopathology do not receive radiotherapy or chemotherapy.

2. Experimental methods

(1) Collecting and storing serum samples:

collecting fasting peripheral blood of a patient in the early morning, placing the fasting peripheral blood in a test tube without anticoagulant, naturally coagulating for 30-60min at room temperature, after blood coagulation, centrifuging for 10min at 2000rpm, carefully sucking supernatant clear serum liquid into a sterile freeze-drying tube, marking, and storing in a refrigerator at-80 ℃ for later use.

3. Experimental and analytical methods

The serum of 580 healthy subjects (normal control group) and 437 cardiac cancer patients (cardiac cancer group) were subjected to qualitative and quantitative determination of two metabolites (xanthhurinic Acid and 2-Hydroxy-2-methylbutyric Acid) according to the UPLC-Q/TOF-MS experimental method described in example 1.

When Xanthus Acid or 2-Hydroxy-2-methylbutanic Acid is independently adopted for cardiac cancer diagnosis, the negative and positive of the sample are judged according to the content of Xanthus Acid or 2-Hydroxy-2-methylbutanic Acid in the serum sample and the optimal cutoff value of the corresponding differential metabolite calculated in the embodiment 2, the content of the differential metabolite in the serum sample is higher than the optimal cutoff value, and the cardiac cancer patient is judged, otherwise, the cardiac cancer patient is judged to be a normal person.

When two metabolic marker combinations of Xanthus Acid and 2-Hydroxy-2-methylbutanic Acid are used for diagnosing cardia cancer, the contents of Xanthus Acid and 2-Hydroxy-2-methylbutanic Acid in a serum sample are substituted into the Logistic regression equation obtained in the example 2, and according to the calculated logit [ p ] value and the Xanthus Acid and 2-Hydroxy-2-methylbutanic Acid combined diagnosis obtained in the example 2, cardia cancer patients and normal persons are distinguished, the negative and positive of the sample are judged, if the logit [ p ] value is higher than the optimal truncation value of 0.468, the cardia cancer is judged, otherwise, the normal person is judged.

3. The result of the detection

TABLE 5 results of cardiac cancer diagnosis using two metabolic markers in combination

The results are shown in Table 5. When the Xanthus Acid or the 2-Hydroxy-2-methylbutanic Acid is independently adopted for cardiac cancer diagnosis, the positive predictive value can reach more than 88 percent, and the negative predictive value can reach more than 89 percent; moreover, when the cardia cancer is diagnosed by combining the Xanthus Acid and the 2-Hydroxy-2-methylbutyric Acid, the positive predictive value and the negative predictive value reach the highest values, namely 92.93 percent and 95.42 percent respectively. Therefore, the combined diagnosis of cardia cancer by using Xanthus Acid and 2-Hydroxy-2-methylbutanic Acid is the best.

Example 4: preparation of cardiac cancer screening kit based on metabolic marker of the invention

Based on the 2 screened metabolic markers related to the cardiac cancer, the cardiac cancer screening kit is designed, and comprises the following components:

standard for markers: at least one of Xanthurenic Acid and 2-Hydroxy-2-methylbutanic Acid, the kit can contain one or 2 metabolic marker standard substances, and the combination can be carried out according to requirements. When the standard substance comprises two metabolic marker substances, the standard substance of the two marker substances can be packaged separately, or the standard substance of the two marker substances can be mixed to be packaged as a mixture.

Internal standard substance: the internal standard substance is L-2-chlorophenylalanine.

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

The use process of the kit is as follows: collecting serum of a subject, freezing and storing the serum in a refrigerator at minus 80 ℃, unfreezing a serum sample in a refrigerator at 4 ℃ before an experiment, taking 50 mu L of the serum sample, then adding 150 mu L of methanol which is 3 times of the volume of the serum sample, uniformly mixing the serum sample by vortex for 30s, putting the mixture into a high-speed centrifuge for centrifugation after uniform mixing, and centrifuging the mixture for 10min at 13000 rpm. Absorbing 75 mu L of each centrifuged supernatant, respectively putting the supernatant into 2 centrifuge tubes with the volume of 1.5ml, blowing the supernatant to dry by using a nitrogen blower, respectively redissolving by using 100 mu L of methanol reconstituted solution containing an internal standard solution (L-2-chlorophenylalanine) after drying, swirling for 30s after redissolution, uniformly mixing, putting the mixture into a high-speed centrifuge for centrifugation for 10min at 13000rpm, absorbing the centrifuged supernatant, and then putting the supernatant into a liquid vial for UPLC-Q/TOF-MS detection. The detection of UPLC-Q/TOF-MS is to analyze the treated serum sample according to the setting method of the UPLC-Q/TOF-MS instrument of the example 1 and to perform quantitative and qualitative analysis on the marker according to the data processing method of the example 1.

When the cardiac cancer detection kit is used, two markers of Xanthus Acid and 2-Hydroxy-2-methylbutanic Acid are suggested to be detected simultaneously, so that the detection efficiency is further improved.

In conclusion, the present invention effectively overcomes the disadvantages of the prior art and has high industrial utilization value. The above-described embodiments are intended to illustrate the substance of the present invention, but are not intended to limit the scope of the present invention. It should be understood by those skilled in the art that various modifications and equivalents may be made to the present invention without departing from the spirit and scope of the invention.

Claims (10)

1. A marker for cardiac cancer screening is 2-Hydroxy-2-methylbutaric acid.

2. Use of a detection reagent for the marker of claim 1 in the preparation of a product for screening cardiac cancer.

3. The use according to claim 2, wherein the test sample of the product is serum.

4. The use of claim 2, wherein the detection reagent is a reagent for detecting the marker in the sample by chromatography, mass spectrometry or a combination of chromatography and mass spectrometry.

5. Use according to claim 4, wherein the chromatography is gas chromatography, liquid chromatography or high performance liquid chromatography.

6. The use of claim 4, wherein the chromatography-mass spectrometry combination is a gas chromatography-mass spectrometry combination, a liquid chromatography-mass spectrometry combination, a high performance liquid chromatography-mass spectrometry combination, a gas chromatography-tandem mass spectrometry combination, a liquid chromatography-tandem mass spectrometry combination, or a high performance liquid chromatography-tandem mass spectrometry combination.

7. A kit for cardiac cancer screening, comprising a detection reagent for detecting the marker of claim 1.

8. The kit of claim 7, wherein the detection reagent is a reagent for detecting the marker in the sample by chromatography-mass spectrometry or a combination of chromatography-mass spectrometry.

9. The kit of claim 8, wherein the chromatography is gas chromatography, liquid chromatography or high performance liquid chromatography; the chromatography-mass spectrometry combined method is a gas chromatography-mass spectrometry combined method, a liquid chromatography-mass spectrometry combined method or a high performance liquid chromatography-mass spectrometry combined method.

10. The kit of claim 9, further comprising a standard for the marker of claim 1.

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