Application of N-acetylneuraminyl glycoprotein as biomarker of coronary atherosclerotic heart disease

Abstract

The invention provides an application of N-acetylneuraminyl glycoprotein as a biological marker of coronary atherosclerotic heart disease. Serum N-acetylneuraminyl glycoprotein is independently related to coronary heart disease risk, the relevance is independent of traditional cardiovascular risk factors, and the serum N-acetylneuraminyl glycoprotein can be used as a biomarker of coronary atherosclerotic heart disease. The N-acetylneuraminyl glycoprotein in the serum sample is quantitatively analyzed through the nuclear magnetic resonance spectrum analysis technology, the level of the N-acetylneuraminyl glycoprotein in the serum can be accurately, rapidly, simply and conveniently quantified, and then early screening is conducted on the coronary heart disease according to the level change of the N-acetylneuraminyl glycoprotein; the detection process is based on a blood sample which is small in trauma and convenient to take, a human body does not need to be exposed to radioactive rays, and the method is safe, reliable and harmless to the human body. The invention further provides application of the equipment or the reagent for detecting the N-acetylneuraminyl glycoprotein in preparation of the system or the kit for detecting the coronary heart disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomarkers, and particularly relates to the application of a composite nuclear magnetic resonance signal derived from an N-acetyl group in an acute phase reaction protein as a metabolomics marker for coronary heart disease. Background Art

[0002] As my country enters an aging society, cardiovascular and cerebrovascular diseases have become common diseases that seriously threaten human health. The "China Cardiovascular Disease Report 2018" shows that in 2016, the mortality rate of cardiovascular and cerebrovascular diseases in my country ranked first, accounting for about 40% of the total mortality. Among them, coronary atherosclerotic heart disease (also known as coronary heart disease), which is caused by coronary artery stenosis, blockage, and subsequent myocardial cell ischemia, hypoxia, and even necrosis due to atherosclerosis, has been ranked first among the top ten causes of death in the world in recent years. According to statistics from the World Health Organization in 2017, about 8.76 million people died of coronary heart disease in the world in 2015, accounting for 15.5% of the total number of deaths. The screening and diagnosis of early coronary heart disease is the key to actively preventing the occurrence of coronary heart disease and reducing mortality.

[0003] Among existing detection technologies, coronary angiography is still the "gold standard" for diagnosing coronary heart disease. However, angiography is an invasive examination, which is complicated to operate, expensive, and has problems such as radiation and contrast agent-related renal damage. Among the non-invasive examination methods, electrocardiogram is the most commonly used, with the advantages of safety, convenience and economy, but its sensitivity and specificity for diagnosing coronary heart disease are low; exercise treadmill test is also a common screening method, but it has exercise-induced risks and a certain false positive and false negative rates; coronary CT can assess the degree of coronary artery stenosis, but there are also risks of radiation and contrast-induced nephropathy, and the image is unclear in cases of arrhythmia or heart failure; radionuclide myocardial perfusion imaging can evaluate the blood perfusion of the corresponding blood supply area, but the technology is not yet recognized, popularized and applied in China, and many medical institutions do not have the conditions for examination; cardiac magnetic resonance imaging has no radiation, but the examination process is complicated, time-consuming, expensive, and has a limited scope of application; the diagnostic value of stress echocardiography has been recognized, but there are certain technical difficulties and exercise-induced risks. Its clinical application in my country is limited and it is difficult to promote.

[0004] In terms of laboratory diagnosis, in order to improve the positive rate of detection and to improve the health economic benefits, researchers have also developed a variety of blood biochemical markers for early screening of coronary heart disease. At present, the most common clinical method is to jointly detect changes in the levels of troponin, creatine kinase isoenzyme, and myoglobin to determine the occurrence of acute coronary heart disease, but because these molecules can be secreted into the blood by various cells throughout the body and can be elevated in a variety of disease environments, the detection specificity and sensitivity of these biochemical indicators are low. In addition, there are also blood lipids and blood sugar in the hematological indicators for early warning of coronary heart disease. However, even if some patients strictly control their blood lipids and blood sugar, they still have the initial onset or recurrence of coronary heart disease, indicating that there may be residual cardiovascular risk. In recent years, a large number of studies have confirmed that chronic inflammation may be one of the main causes of residual risk. Therefore, C-reaction protein (CRP), as a classic inflammatory marker, has gradually been used to assess the risk of residual cardiovascular inflammation, but CRP has large inter-individual and intra-individual variability, which limits its clinical application. Considering the serious threat of coronary heart disease, it is urgent to use body fluid samples that are less invasive and easy to obtain to explore new screening markers to help with the early screening of coronary heart disease.

[0005] Metabolomics is the study of the dynamic changes of metabolites in different tissues, cells, and body fluids in the body after an organism is subjected to physiological and pathological stimulations such as gene mutations or environmental disturbances. As the most downstream of systems biology, it is closest to the functional state and disease phenotype of the organism. Among them, metabolomics based on nuclear magnetic resonance spectroscopy (NMR) has become a valuable technical application due to its non-invasiveness, short detection time, high reproducibility, and absolute quantification. There are fewer sample processing steps before NMR spectroscopy analysis, which avoids biologically irrelevant biases and interference. In addition, NMR spectroscopy is a non-destructive technology that can perform single-dimensional or multi-dimensional detection on a single sample and obtain comprehensive parameter information in a short time. In the past 20 years, NMR spectroscopy metabolomics technology has been widely used in the study of various disease backgrounds, and has also provided a new means for finding biomarkers for coronary heart disease.

[0006] Blood N-acetylneuraminyl-glycoproteins (NAG2) were first reported by Bell et al. in 1987. They are a composite nuclear magnetic resonance signal derived from the N-acetyl group (δ2.08ppm) containing the N-acetylneuraminic acid sugar side chain in acute phase proteins. Acute phase proteins are a class of proteins whose blood concentration changes significantly when the body is exposed to harmful stimuli, including α1-antitrypsin, α1-antichymotrypsin, α1-acid glycoprotein, transferrin, haptoglobin, etc. N-acetylneuraminyl-glycoproteins originate from the sialic acid part of these acute phase proteins, and changes in their signal levels represent changes in the concentration of a series of glycoproteins. Studies have shown that increased blood N-acetylneuraminyl-glycoprotein levels not only indicate acute inflammatory reactions, but can also be used as a biomarker for chronic inflammatory diseases such as rheumatoid arthritis, obesity, and polycystic ovary syndrome. A recent study also found that serum N-acetylneuraminyl-glycoprotein can be used as a predictive marker for all-cause mortality in patients with heart failure. However, no studies have confirmed whether there is a correlation between serum N-acetylneuraminyl glycoprotein and the occurrence of coronary heart disease, which is very important for determining the severity of the disease and formulating the next treatment plan.

[0007] Therefore, exploring the diagnostic value of serum N-acetylneuraminyl glycoprotein for coronary heart disease based on nuclear magnetic resonance spectroscopy metabolomics technology is of great significance for early screening and treatment of coronary heart disease and improving patient survival rate. Summary of the invention

[0008] The purpose of the present invention is to provide an application of N-acetyl groups in acute phase reaction proteins as serum metabolomics markers for the diagnosis of coronary heart disease. By detecting 180 nuclear magnetic resonance spectroscopy serum metabolites in 6263 residents of Changfeng Community in Shanghai, it was found that serum N-acetylneuraminyl glycoprotein had the strongest correlation with the risk of carotid atherosclerosis among all metabolites, and this association was independent of traditional metabolic risk factors. Combining serum N-acetylneuraminyl glycoprotein with Framingham risk score can significantly improve the evaluation efficiency of carotid atherosclerosis. Therefore, serum N-acetylneuraminyl glycoprotein can become a new biomarker for assessing the risk of carotid atherosclerosis.

[0009] Acute phase reaction protein (APRP) refers to: when the human body is invaded by tissue damage such as infection, autoimmune disease (such as trauma, surgery, myocardial infarction, tumor, etc.), inflammation is induced, and cells such as monocytes and macrophages release emergency reactive cytokines, which then circulate through the blood and stimulate liver cells to produce C-reactive protein, a1-antitrypsin, a1-acid glycoprotein, haptoglobin, ceruloplasmin, C3, C4, fibrinogen and serum amyloid A, etc., which significantly increase their plasma concentrations, while the concentrations of plasma prealbumin, albumin and transferrin decrease accordingly. This inflammatory reaction process is called acute phase reaction. The proteins that appear in this process are collectively called acute phase reaction proteins.

[0010] To achieve the above objectives and other related objectives, the first aspect of the present invention is the use of N-acetylneuraminyl-glycoproteins (NAG2) as a biomarker for coronary atherosclerotic heart disease.

[0011] N-Acetylneuraminic Acid (CAS NO.131-48-6) is a nine-carbon carbohydrate α-keto acid that is present in large quantities in human and mammalian tissues and several body fluids including serum, cerebrospinal fluid, saliva, amniotic fluid and breast milk. Neu5Ac is a sugar chain component of almost all glycoproteins, glycolipids and glycopeptides in the body. These molecules play an important role in cell recognition, cell localization and cell adhesion, as well as in immune responses such as blood protein half-life regulation and toxicity neutralization. It constitutes the terminal sugar chain of cell surface glycoproteins and plays an important role in cell recognition, cell localization and cell adhesion; it constitutes the terminal sugar chain of cell membrane surface glycoproteins and plays an important role in tumor growth and metastasis; it constitutes the terminal sugar chain of sialylated glycoproteins in serum and regulates the half-life of these proteins and possible related physiological functions; it constitutes the terminal sugar chain of gangliosides in the human brain and is the transmitter of gangliosides.

[0012] Serum N-acetylneuraminyl glycoprotein is a type of glycoprotein present in serum or blood. The protein part of the protein has differences in the primary structure, while the sugar part is all N-acetylneuraminyl. This application shows, through a study of serum samples from a large population, that serum N-acetylneuraminyl glycoprotein has the strongest correlation with the risk of carotid atherosclerosis among all metabolites, and this association is independent of traditional metabolic risk factors. Therefore, it can be used as a biomarker for coronary atherosclerotic heart disease. Quantitative detection of its expression in serum or blood can determine whether the subject suffers from coronary atherosclerotic heart disease with high specificity and sensitivity.

[0013] As certain embodiments of the present invention, the N-acetylneuraminyl glycoprotein is derived from body fluids. As certain embodiments of the present invention, the N-acetylneuraminyl glycoprotein is derived from a blood sample or a serum sample.

[0014] The second aspect of the present invention is to provide the use of a device for detecting N-acetylneuraminyl glycoprotein in preparing a system for detecting the risk of coronary atherosclerotic heart disease or predicting the risk of cardiovascular death within 5 years.

[0015] The inventors found through population sample testing that serum NAG2 is independently correlated with the risk of coronary heart disease, and this association is independent of traditional cardiovascular risk factors such as age, smoking, hypertension, hyperglycemia, and hyperlipidemia.

[0016] The device for detecting N-acetylneuraminyl glycoprotein refers to a device for detecting N-acetylneuraminyl in N-acetylneuraminyl glycoprotein, or detecting N-acetyl groups in N-acetylneuraminyl glycoprotein, which can be a nuclear magnetic resonance spectroscopy or a liquid chromatography-mass spectrometer.

[0017] The principle of nuclear magnetic resonance spectroscopy (NMR) analysis is the physical process that the spin energy level of atomic nuclei with non-zero magnetic moments splits under the action of an external magnetic field and resonates to absorb radio frequency radiation of a certain frequency. It can be used to determine the number, type and relative position of certain atoms in a molecule. The nuclear magnetic resonance spectrum can directly provide various chemical or physical states of a specific atom in a sample, and obtain their respective quantitative data, which do not require calibration with standard substances. The peak area is proportional to the number of atoms that produce the area.

[0018] As certain embodiments of the present invention, the subject of the application is an adult.

[0019] The subjects of the application may include different groups of people, such as healthy people, people with coronary artery stenosis but not diagnosed with coronary heart disease, and subjects suspected of coronary heart disease. Serum samples of the subjects to be tested are collected, and the concentration of biomarkers in the body fluid samples is quantitatively detected by a nuclear magnetic resonance spectrometer.

[0020] As certain embodiments of the present invention, the application targets Chinese people.

[0021] The inventors found through population sample testing that the systemic inflammation level reflected by the increased serum NAG2 level partially explains the risk of coronary heart disease in Chinese people. Serum NAG2 can be used as a new biomarker for screening the risk of coronary heart disease.

[0022] As certain embodiments of the present invention, the sample for detection is taken from the body fluid of the subject of the application. As certain embodiments of the present invention, the sample for detection is taken from the blood or serum of the subject of the application.

[0023] As certain embodiments of the present invention, the device for detecting N-acetylneuraminyl glycoprotein (NAG2) is a nuclear magnetic resonance spectroscopy device or a liquid chromatography-mass spectrometry device.

[0024] As certain embodiments of the present invention, the equipment for the nuclear magnetic resonance spectroscopy analysis is a Bruker AVANCEⅢ600 nuclear magnetic resonance spectrometer, and the detection parameters include an experimental temperature of 310K, a sampling point number of 98K, a cumulative number of 32 times, a spectral width of 20ppm, and a line width factor of 0.3Hz.

[0025] As certain embodiments of the present invention, the specific procedure of the nuclear magnetic resonance spectroscopy analysis is as follows: 1D 1H nuclear magnetic resonance spectroscopy experiment and diffusion weighted experiment are selected, the 1H nuclear magnetic resonance spectroscopy experiment uses the NOESYGPPR1D pulse sequence to obtain the full metabolite spectrum signal, and the diffusion weighted experiment uses the LEDBPGPPR2S1D pulse sequence, and the small molecule signal is attenuated by optimizing the diffusion delay time parameter, thereby retaining the macromolecular signal; the preprocessing of the nuclear magnetic resonance spectrum is realized in the Topspin3.6.0 software; based on MATLAB R2022b (Mathworks Inc.) software, the fitting of the metabolite signal uses the Gaussian-Lorentzian mixed line shape as the main line shape, and in the case of multiple spectral lines, the measured line shape is reconstructed by the sum of the Gaussian-Lorentzian lines, and the Lorentzian and Gaussian percentages of each signal are calculated. The locally optimized first-order polynomial is used to roughly estimate the baseline contribution, and the offset and slope are added to the multiple line shapes as fitting parameters, thereby quantitatively obtaining the N-acetylneuraminyl glycoprotein level from the nuclear magnetic resonance spectrum; 1D The detection limit of 1H NMR spectroscopy is at the μM level.

[0026] As certain embodiments of the present invention, the preprocessing of the NMR spectrum includes FID Fourier transform, phase adjustment, baseline correction, chemical shift calibration, and spectrum peak alignment.

[0027] As certain embodiments of the present invention, the detection limit of the 1D 1H nuclear magnetic resonance spectrum is 1-1000 μM.

[0028] The detection limit of the 1D 1H NMR spectrum is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 μM.

[0029] The detection limit of 1D 1H NMR spectroscopy is usually at the μM level, but the complex coupled splitting, residual solvents, signal overlap, etc. in biological samples reduce the signal-to-noise ratio, resulting in a too high detection limit for specific signals (metabolite profiling is often above 100 μM).

[0030] As certain embodiments of the present invention, the quality control measures of the nuclear magnetic resonance spectroscopy analysis include testing standard substances every day, testing mixed quality control (QC) biological samples prepared under the same conditions before testing each batch of biological samples, evaluating the reproducibility of the detection instrument based on the relative standard deviation (RSD) of the measurement results, and the reproducibility RSD of the detection instrument is <20%; adjusting the preparation quantity of the samples daily according to the temperature equilibration time of the samples and the instrument tuning time to ensure that each sample obtains nuclear magnetic resonance spectroscopy data within 24 hours after preparation, thereby reducing biologically irrelevant deviations and interferences; before collecting the nuclear magnetic resonance spectroscopy spectrum, a deuterated methanol sample is used to calibrate the experimental temperature, a mixed standard substance reference sample is used for concentration quantitative calibration, and a sucrose reference sample is used for solvent peak suppression effect evaluation, thereby ensuring the stability of the detection environment and equipment.

[0031] As certain embodiments of the present invention, the sample used for the nuclear magnetic resonance spectroscopy analysis is serum diluted with an equal volume of phosphate buffer, and the concentration of the phosphate buffer is 0.085M.

[0032] As certain embodiments of the present invention, the system includes a data analysis module for calculating the Gensini score of the sample to be tested based on the level of N-acetylneuraminyl glycoprotein in the sample to be tested using a regression model, thereby assessing the risk of coronary atherosclerotic heart disease or predicting the 5-year risk of cardiovascular death.

[0033] Preferably, the regression model is constructed and trained using the N-acetylneuraminyl glycoprotein test values ​​and Gensini scores of known samples; further preferably, the regression model is a restricted cubic spline (RCS) regression model; further preferably, the regression model also includes a correction term, and the correction term is selected from one or more of age, gender, smoking, body mass index (BMI), systolic blood pressure (SBP), fasting blood glucose (FPG), serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), brain natriuretic peptide (BNP), and inflammatory markers; more preferably, a multifactor Logistics regression model is used to correct one or more of age, gender, smoking, body mass index (BMI), systolic blood pressure (SBP), fasting blood glucose (FPG), serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), brain natriuretic peptide (BNP), and inflammatory markers.

[0034] The Gensini score quantitatively evaluates the degree of stenosis of each coronary artery lesion based on the results of coronary angiography. The degree of stenosis is based on the most severe part. Stenosis diameter <25% is scored as 1 point, 25%≤diameter<50% is scored as 2 points, 50%≤diameter<75% is scored as 4 points, 75%≤diameter<90% is scored as 8 points, 90%≤diameter<99% is scored as 16 points, and ≥99% is scored as 32 points.

[0035] The restricted cubic spline (RCS) regression model is a statistical method to study the nonlinear relationship between the independent variable x and the dependent variable y.

[0036] The multi-factor logistics regression model is a scientific statistical method that considers the impact of multiple factors on the results to obtain more accurate prediction results. This model can also be used to explore the impact of different factors on the results.

[0037] As certain embodiments of the present invention, the inflammatory marker is selected from white blood cell count and high-sensitivity C-reactive protein.

[0038] The third invention of the present invention is to provide the use of a reagent for detecting N-acetylneuraminyl glycoprotein in the preparation of a kit for detecting the risk of coronary atherosclerotic heart disease or predicting the risk of cardiovascular death in 5 years.

[0039] As certain embodiments of the present invention, the subject of the application is an adult.

[0040] As certain embodiments of the present invention, the application targets Chinese people.

[0041] As certain embodiments of the present invention, the sample for detection is taken from the body fluid of the subject of the application. As certain embodiments of the present invention, the sample for detection is taken from the blood or serum of the subject of the application.

[0042] As certain embodiments of the present invention, the reagent for detecting N-acetylneuraminyl glycoprotein is a reagent for detecting N-acetylneuraminic acid in N-acetylneuraminyl glycoprotein, or a reagent for detecting N-acetyl group in N-acetylneuraminyl glycoprotein.

[0043] As certain embodiments of the present invention, the application is used in combination with other equipment, and the other equipment is selected from one or more of the following: coronary angiography, electrocardiogram, echocardiography, exercise treadmill test, coronary CT angiography, radionuclide myocardial perfusion imaging, and cardiac magnetic resonance imaging.

[0044] Since NAG2 can also be used as an inflammatory marker and has a certain correlation with other chronic diseases, the results of coronary heart disease screening can be made more reliable by combining serum NAG2 levels with traditional cardiovascular risk factors.

[0045] The fourth aspect of the present invention is to provide the use of a reagent for detecting N-acetylneuraminyl glycoprotein in the preparation of a diagnostic agent, wherein the diagnostic agent is used for diagnosing coronary atherosclerotic heart disease.

[0046] The inventors have found through population sample testing that serum NAG2 is independently associated with the risk of coronary heart disease, and this association is independent of traditional cardiovascular risk factors such as age, smoking, hypertension, hyperglycemia, hyperlipidemia, etc. Based on this, N-acetylneuraminyl glycoprotein in serum or blood samples is considered to be a new biomarker for coronary atherosclerotic heart disease. The diagnostic agent provided by the present invention diagnoses whether the test subject suffers from coronary atherosclerotic heart disease by detecting N-acetylneuraminyl glycoprotein in serum or blood samples.

[0047] The fifth aspect of the present invention is to provide the use of a device or reagent for detecting N-acetylneuraminyl glycoprotein in preparing a system or a kit, wherein the system or the kit is used to screen drugs for treating coronary atherosclerotic heart disease.

[0048] The present application provides an application for screening drugs for treating coronary atherosclerotic heart disease by detecting the presence or level of N-acetylneuraminyl glycoprotein in the blood or serum samples of patients after taking drugs for treating coronary atherosclerotic heart disease to verify the effectiveness of the drugs taken by the patients.

[0049] The sixth aspect of the present invention is to provide a device for detecting the risk of coronary atherosclerotic heart disease or predicting the 5-year cardiovascular death risk, the device comprising at least the following modules: an N-acetylneuraminylglycoprotein content detection module, used to detect the N-acetylneuraminylglycoprotein content in the serum sample or blood sample of the subject, and to detect the N-acetylneuraminylglycoprotein or N-acetyl group of the N-acetylneuraminylglycoprotein in the serum sample or blood sample of the subject to determine the total content of the N-acetylneuraminylglycoprotein; a data analysis module, used to calculate the Gensini score of the sample to be tested based on the N-acetylneuraminylglycoprotein level in the sample to be tested using a regression model, thereby evaluating the risk of coronary atherosclerotic heart disease or predicting the 5-year cardiovascular death risk.

[0050] Preferably, the regression model is constructed and trained using the N-acetylneuraminyl glycoprotein test value and Gensini score of known samples; further preferably, the regression model is a restricted cubic spline (RCS) regression model; further preferably, the regression model also includes a correction term, and the correction term is selected from one or more of age, gender, smoking, body mass index (BMI), systolic blood pressure (SBP), fasting blood glucose (FPG), serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), brain natriuretic peptide (BNP), and inflammatory markers; more preferably, a multifactorial Logistics regression model is used to correct one or more of age, gender, age, gender, smoking, body mass index (BMI), systolic blood pressure (SBP), fasting blood glucose (FPG), serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), brain natriuretic peptide (BNP), and inflammatory markers.

[0051] The fifth aspect of the present invention is to provide an electronic terminal, which includes: a processor, a memory, and a communicator; the memory is used to store a computer program; preferably, the computer program runs a restricted cubic spline (RCS) regression model and a multifactor logistics regression model; the communicator is used to communicate with an external device to receive the detected N-acetylneuraminyl glycoprotein content data; the processor is used to execute the computer program stored in the memory, so that the electronic terminal analyzes the N-acetylneuraminyl glycoprotein level after correcting the influence of the influencing factors, obtains the Gensini score of the sample to be tested, and thus evaluates the risk of coronary atherosclerotic heart disease or predicts the 5-year cardiovascular death risk.

[0052] As described above, the use of the N-acetylneuraminyl glycoprotein of the present invention as a biomarker for coronary atherosclerotic heart disease has the following beneficial effects:

[0053] The present invention is based on the nuclear magnetic resonance spectroscopy metabolomics analysis platform, and analyzed more than 4,000 participants from the GRAND cohort. It was found that there were significant differences in serum N-acetylneuraminyl glycoprotein levels between patients with coronary heart disease and healthy people. The serum N-acetylneuraminyl glycoprotein levels in patients with coronary heart disease were significantly increased, and showed an independent linear relationship with the Gensini score that reflects the degree of coronary artery lumen stenosis, suggesting that serum N-acetylneuraminyl glycoprotein can be used as a new and effective biomarker for screening coronary heart disease.

[0054] Coronary heart disease is a chronic inflammatory vascular disease. Its occurrence and development are the result of the interaction of multiple genetic and environmental factors. It is a complex metabolic disease. The diagnosis of coronary heart disease in the prior art mainly relies on coronary angiography. However, due to the high price and invasiveness of angiography, many patients hesitate to undergo the examination. Therefore, the screening and diagnosis of early coronary heart disease of the present invention, especially the body fluid examination with low trauma and convenient sampling, is of great significance for preventing disease progression and improving patient survival rate.

[0055] Given that body fluid extraction is simple and convenient, extracting venous blood from the test subject to analyze the level of serum N-acetylneuraminyl glycoprotein can quickly reflect the onset of coronary heart disease, thus providing a new detection direction for the diagnosis of coronary heart disease. In actual clinical applications, it can also be combined with other existing detection methods to improve the accuracy of coronary heart disease diagnosis and further improve the diagnosis rate of patients.

[0056] Serum N-acetylneuraminyl glycoprotein is a parameter measured based on nuclear magnetic resonance spectroscopy. Nuclear magnetic resonance spectroscopy has the characteristics of strong stability and good repeatability, so the individual variability of the test results is small.

[0057] Serum N-acetylneuraminyl glycoprotein is a composite nuclear magnetic resonance signal. Its level changes represent the concentration changes of a series of glycoproteins rather than a specific protein molecule. Therefore, it can better capture the fluctuations of systemic inflammation levels.

[0058] The detection time is short, and the serum N-acetylneuraminyl glycoprotein level can be quantified quickly and easily, thereby performing early screening for coronary heart disease based on changes in its level;

[0059] The detection process is based on blood samples that are less invasive and easy to obtain. The human body does not need to be exposed to radiation. It has the advantages of being safe, reliable, and harmless to the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1The distribution of serum N-acetylneuraminyl glycoprotein in different populations of the present invention is shown, the red and blue dotted lines represent the middle levels of patients with coronary heart disease and controls without coronary heart disease, respectively, and the concentration levels are marked in the top area;

[0061] Figure 2 Shown is the dose-response relationship between serum N-acetylneuraminic glycoprotein level and Gensini score of the present invention, red shading represents 95% confidence interval, Poverall and Pnonlinear reflect the statistical association and nonlinear association between serum N-acetylneuraminic glycoprotein and Gensini score, respectively;

[0062] Figure 3 The ORs (95% CI) of coronary heart disease for each 1 standard deviation increase in serum N-acetylneuraminyl glycoprotein level after stepwise adjustment of traditional cardiovascular risk factors using the multivariate logistic regression model of the present invention are shown;

[0063] Figure 4 Shown are the ORs (95% CI) of coronary heart disease for each 1 standard deviation increase in serum N-acetylneuraminyl glycoprotein levels in different subgroups of the present invention;

[0064] Figure 5 It shows the diagnostic efficacy of serum N-acetylneuraminyl glycoprotein, traditional cardiovascular risk factors and their combination for coronary heart disease of the present invention;

[0065] Figure 6 It is shown that the elevated serum N-acetylneuraminyl glycoprotein of the present invention predicts an elevated risk of cardiovascular death within 5 years. DETAILED DESCRIPTION

[0066] The present invention is further described below in conjunction with specific examples, and it should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0067] It should be understood that the terms used in the examples of the present invention are intended to describe specific embodiments rather than to limit the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated, the singular forms "a", "an" and "the" include plural forms. When the examples give numerical ranges, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected.

[0068] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, any methods, equipment, and materials of the prior art that are similar or equivalent to the methods, equipment, and materials in the embodiments of the present invention may be used to implement the present invention, based on the prior art mastery of those skilled in the art and the description of the present invention.

[0069] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the technical field and related fields.

[0070] Materials and Methods

[0071] Study population and ethical statement

[0072] The GRAND study is a multicenter, prospective, case-control study. All subjects were from the GRAND study (2894 men and 1461 women, with an average age of 59.4 years and a BMI of 24.8 kg / m 2 ). There were 2542 patients in the CHD group, who were selected from patients with CHD who were included in the GRAND cohort study between May 2017 and May 2018. All patients with CHD underwent coronary angiography. CHD was diagnosed if at least one major epicardial coronary artery (left anterior descending artery, left circumflex artery, or right coronary artery) had a stenosis of ≥50% under coronary angiography. There were 1813 patients in the control group, who were selected from subjects without CHD who were included in the GRAND cohort study between May 2017 and May 2018.

[0073] This study was conducted in accordance with the Declaration of Helsinki and approved by the Research Ethics Committee of Zhongshan Hospital of Fudan University. Written informed consent was obtained from all participants.

[0074] Questionnaire

[0075] A uniformly designed epidemiological questionnaire was used, and qualified staff conducted face-to-face interviews with participants. The content included name, gender, age, ethnicity, marital status, type of work, home address and contact information, personal lifestyle habits such as smoking and drinking, history of hypertension, diabetes, dyslipidemia, family history of cardiovascular disease, and medication history.

[0076] Physical examination

[0077] When measuring weight, the subject takes off his shoes and hat, and the result is accurate to 0.1kg; when measuring height, the subject stands upright, with his back straight and his feet together, and the result is accurate to 0.1cm; the body mass index (BMI) is calculated according to the formula = weight / height (kg / m 2 ).

[0078] Blood pressure was measured in the sitting position. The subject was asked to relax and sit quietly for 5-10 minutes. The subject's right upper arm was placed on the examination table, flush with the heart height, and the measurement was performed using an electronic sphygmomanometer (OMRON Model HEM-752FUZZY, Omron Co., Dalian, China) calibrated from a traditional mercury sphygmomanometer. The measurement was repeated 3 times, with an interval of at least 1 minute each time. The average of the 3 measurements was recorded and calculated.

[0079] Biochemical testing

[0080] The subjects fasted overnight for at least 10 hours, and 10 ml of fasting peripheral blood was collected intravenously in the morning of the next day and placed in a serum tube. The biochemical parameters of the subjects were detected using an automatic biochemical analyzer (HITACHI 7600, Tokyo, Japan). Fasting plasma glucose (FPG) was measured by glucose oxidase method, and serum total cholesterol (TC), triglyceride (TG), and high-density lipoprotein cholesterol (HDL-C) were measured by oxidase method. LDL-C was calculated according to Friedewald formula = (TC-HDL-C)-TG / 2.2.

[0081] Coronary artery stenosis score

[0082] All participants underwent coronary angiography, and each coronary artery was scored according to the degree of luminal stenosis (Gensini scores for coronary artery stenosis of 25%, 50%, 75%, 90%, 99%, and complete occlusion were 1, 2, 4, 8, 16, and 32 points, respectively). The total Gensini score for each participant was calculated based on the weighting coefficient of different coronary arteries.

[0083] Serum metabolomics detection based on nuclear magnetic resonance spectroscopy

[0084] The detection equipment is a Bruker AVANCEⅢ600 nuclear magnetic resonance spectrometer, equipped with a SampleJet automatic sampler with a temperature control system (to keep the sample at 4°C before measurement), a BBI probe and a high-order shim system.

[0085] The detection parameters include experimental temperature of 310K, sampling points of 98K, accumulation times of 32 times, spectrum width of 20ppm, and line width factor of 0.3Hz.

[0086] The detection method is conventional 1D 1 H NMR spectroscopy and diffusion-weighted experiments. The NOESYGPPR1D pulse sequence was used to detect the full metabolite spectrum signal; the LEDBPGPPR2S1D pulse sequence was used to attenuate the small molecule signal and retain the macromolecule signal by optimizing the diffusion delay time and other parameters. The preprocessing of the NMR spectrum (including FID Fourier transform, phase adjustment, baseline correction, chemical shift calibration, and peak alignment) was implemented in Topspin (3.6.0) software; based on MATLABR2022b (Mathworks Inc.) software, the fitting of the metabolite signal used a Gaussian-Lorentzian mixed line shape as the main line shape. In the case of multiple spectral lines, the measured line shape was reconstructed by the sum of Gaussian-Lorentzian lines, and the Lorentzian and Gaussian percentages of each signal were calculated. The locally optimized first-order polynomial was used to roughly estimate the baseline contribution, and the offset and slope were added to the multiple line shapes as fitting parameters to quantitatively obtain the N-acetylneuraminyl glycoprotein level from the NMR spectrum. The detection limit of 1D 1H NMR spectroscopy is usually at the μM level, but the complex coupled splitting, residual solvents, signal overlap, etc. in biological samples reduce the signal-to-noise ratio, resulting in a too high detection limit for specific signals (metabolite profiling is often above 100 μM).

[0087] Quality control measures include regular testing of standard substances (regularity can be daily), testing of mixed quality control (QC) biological samples prepared under the same conditions before testing each batch of biological samples, and evaluating the reproducibility of the testing instrument based on the relative standard deviation (RSD) of the measurement results (RSD < 20%); adjusting the number of samples prepared daily according to the temperature equilibration time of the samples and the instrument tuning time (the number of samples to be prepared each day is calculated based on the longest time required for each sample from injection to the end of testing within 24 hours) to ensure that spectral data is obtained for each sample within 24 hours after preparation, thereby reducing biologically irrelevant deviations and interferences; calibrating the experimental temperature with a deuterated methanol sample before spectral acquisition (detecting the experimental temperature through a program, and adjusting the corresponding parameters to control the experimental temperature within ±0.05°C), performing quantitative concentration calibration with a mixed standard substance reference sample, and evaluating the solvent peak suppression effect with a sucrose reference sample, thereby ensuring the stability of the testing environment and equipment.

[0088] Serum tubes were centrifuged at 3000 rpm for 15 minutes to separate serum, and 0.5 ml of serum was transferred to the nuclear magnetic resonance spectroscopy detection platform at low temperature. Phosphate buffer (0.085 M containing 10% D2O) was prepared, and the pH was measured and adjusted to 7.38-7.42. Serum samples were centrifuged at 2700 × g for 10 minutes, and 350 μL of supernatant was mixed with 350 μL of 0.085 M phosphate buffer. 600 ul of the mixture was transferred to a 5 mm nuclear magnetic resonance spectroscopy tube and placed in a SampleJet autosampler. The nuclear magnetic resonance spectrometer automatically collected spectral data for each sample in turn through the set program. The serum N-acetylneuraminyl glycoprotein level was relatively quantified from the diffusion-weighted spectrum.

[0089] Statistical methods

[0090] Two-sided P value < 0.05 was set as the significance threshold. Descriptive statistics were used according to the type of variables. Student's t test was used to compare the differences in serum N-acetylneuraminyl glycoprotein levels between patients with coronary heart disease and those without coronary heart disease.

[0091] The dose-response relationship between serum N-acetylneuraminic acid protein level and Gensini score was analyzed using restricted cubic spline (RCS) regression model, and age, sex, smoking, BMI, systolic blood pressure, FPG, TG, LDL-C, and HDL-C were adjusted in the model. The correlation between serum N-acetylneuraminic acid protein level and coronary artery disease (CAD) was analyzed using multivariate logistics regression model after adjusting for age, sex, smoking, BMI, SBP, FPG, TC, TG, HDL-C, and inflammatory markers (white blood cell count and high-sensitivity C-reactive protein).

[0092] The population was stratified according to age (<65 or ≥65 years old), sex (female or male), BMI (<24 or 24-28 or ≥28 kg / m2), hypertension (yes or no), diabetes (yes or no), dyslipidemia (yes or no), and metabolic health (yes or no). Logistic regression model was used to analyze the relationship between serum N-acetylneuraminyl glycoprotein level and coronary heart disease in the stratified population. At the same time, the interaction terms between N-acetylneuraminyl glycoprotein and these traditional risk factors were introduced into the model to analyze the interaction.

[0093] The receiver operating characteristic (ROC) curve was used to analyze the diagnostic performance of serum N-acetylneuraminyl glycoprotein, traditional cardiovascular risk factors and their combination for coronary heart disease. The DeLong test was used to compare the area under the ROC curve (AUC). The larger the AUC, the greater the diagnostic efficacy of the indicator, that is, the higher the diagnostic accuracy.

[0094] Example 1 Differences in serum N-acetylneuraminyl glycoprotein levels between people with and without coronary heart disease

[0095] Serum N-acetylneuraminic glycoprotein showed an approximately normal distribution in both patients with and without coronary heart disease. The average level of serum N-acetylneuraminic glycoprotein in patients with coronary heart disease was 0.85 peak areas, while the level of N-acetylneuraminic glycoprotein in patients without coronary heart disease was 0.83 peak areas (P<0.001). The serum N-acetylneuraminic glycoprotein in patients with coronary heart disease was significantly higher. The analysis results are shown in Figure 1 . Figure 1 The distribution of serum N-acetylneuraminyl glycoprotein in different populations is shown. The red and blue dashed lines represent the intermediate levels of patients with coronary heart disease and controls without coronary heart disease, respectively, and the concentration levels are marked in the top area.

[0096] Example 2 Association between serum N-acetylneuraminyl glycoprotein level and coronary heart disease

[0097] In the general population, serum N-acetylneuraminyl glycoprotein levels were linearly correlated with Gensini scores (Poverall = 0.038, Pnonlinear = 0.474). The analysis results are shown in Figure 2 Serum N-acetylneuraminyl glycoprotein level was independently associated with the risk of coronary heart disease (OR, 1.48; 95% CI, 1.27-1.73). The analysis results are shown in Figure 3 . Figure 2 The dose-response relationship between serum N-acetylneuraminic acid protein level and Gensini score is shown. The red shading represents the 95% confidence interval. Poverall and Pnonlinear reflect the statistical association and nonlinear association between serum N-acetylneuraminic acid protein and Gensini score, respectively. Figure 3 The ORs (95% CI) of CHD per 1 SD increase in serum N-acetylneuraminyl glycoprotein level after stepwise adjustment for traditional cardiovascular risk factors using multivariable logistic regression models are shown.

[0098] We further divided the two study populations into subgroups based on age, gender, BMI, and metabolic status. The analysis found that except for patients over 65 years old or with diabetes, serum N-acetylneuraminyl glycoprotein levels were significantly associated with the risk of coronary heart disease (ORs: 1.25-1.83, P values ​​< 0.001). There was no interaction between serum N-acetylneuraminyl glycoprotein and these traditional cardiovascular risk factors (interaction P values ​​were all > 0.05). The analysis results are shown in Figure 4 . Figure 4 The ORs (95% CI) of CHD for each 1 standard deviation increase in serum N-acetylneuraminyl glycoprotein level in different subgroups are shown.

[0099] Example 3 Serum N-acetylneuraminyl glycoprotein improves the diagnostic efficacy of coronary heart disease

[0100] The clinical score of each participant was calculated based on the traditional cardiovascular risk factors (including age, gender, FBG, systolic blood pressure, LDL-C, TG) using a logistic regression model. The ROC curve showed that the clinical score was significantly correlated with coronary heart disease (AUROC, 0.84; 95% CI, 0.80-0.86). Serum N-acetylneuraminyl glycoprotein levels can independently identify patients with coronary heart disease, and combined with traditional risk factors can further improve the accuracy of identifying the risk of coronary heart disease (AUROC, 0.89; 95% CI, 0.88-0.90) (P<0.001). The analysis results are shown in Figure 5 . Figure 5 The results showed that serum N-acetylneuraminyl glycoprotein, traditional cardiovascular risk factors and their combination have diagnostic efficacy for coronary heart disease. The optimal cut-off point was obtained by maximizing the Youden index. The clinical data combined with the NAG2 model had a sensitivity of 80% and a specificity of 82% for diagnosing cardiovascular disease, while the NAG2 alone had a sensitivity of 53% and a specificity of 70%.

[0101] Example 4: Elevated serum N-acetylneuraminyl glycoprotein predicts increased risk of cardiovascular death in 5 years

[0102] In the validation population (6044 people), N-acetylneuraminyl glycoprotein was shown to independently predict the risk of cardiovascular death. Combined with correction for different risk factors, the results are shown in Table 1:

[0103] Table 1 NAG2 and 5-year all-cause and cause-related mortality risk in the validation population (n=6,044)

[0104]

[0105] Figure 6 The present invention also shows that elevated serum N-acetylneuraminyl glycoprotein predicts an elevated risk of cardiovascular death within 5 years.

[0106] The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and evolutions of the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. Application of N-acetylneuraminyl-glycoproteins (NAG2) as a biomarker for coronary atherosclerotic heart disease.

2. The use according to claim 1, characterized in that: The N-acetylneuraminyl glycoprotein is derived from a body fluid, preferably, the N-acetylneuraminyl glycoprotein is derived from a blood sample or a serum sample.

3. Use of a device for detecting N-acetylneuraminyl glycoprotein in the preparation of a system for detecting the risk of coronary atherosclerotic heart disease or predicting the 5-year risk of cardiovascular death.

4. The use according to claim 3, characterized in that: The application is targeted at the adult population; Or, the target of the application is Chinese; Alternatively, the sample to be tested is obtained from a body fluid of the subject of the application; preferably, the sample to be tested is obtained from the blood or serum of the subject of the application.

5. The use according to any one of claims 3 to 4, characterized in that: The device for detecting N-acetylneuraminyl glycoprotein is a nuclear magnetic resonance (NMR) spectroscopy (LC-MS) analysis device or a liquid chromatography-mass spectrometry device; Preferably, the equipment for the nuclear magnetic resonance spectroscopy analysis is a Bruker AVANCEⅢ600 nuclear magnetic resonance spectrometer, and the detection parameters include an experimental temperature of 310K, a sampling point number of 98K, a cumulative number of 32 times, a spectrum width of 20ppm, and a line width factor of 0.3Hz.

6. The use according to claim 5, characterized in that: The specific procedure of the nuclear magnetic resonance spectroscopy analysis is: Choose 1D 1 H NMR spectroscopy experiments and diffusion-weighted experiments, 1 The H NMR spectroscopy experiment used the NOESYGPPR1D pulse sequence to obtain the full metabolite spectrum signal, and the diffusion-weighted experiment used the LEDBPGPPR2S1D pulse sequence to attenuate the small molecule signal by optimizing the diffusion delay time parameter, thereby retaining the large molecule signal; The preprocessing of the NMR spectrum is implemented in Topspin3.6.0 software; based on MATLAB R2022b (Mathworks Inc.) software, the fitting of the metabolite signal adopts a Gaussian-Lorentzian mixed line shape as the main line shape. In the case of multiple spectral lines, the measured line shape is reconstructed by the sum of Gaussian-Lorentzian lines, and the Lorentzian and Gaussian percentages of each signal are calculated. The locally optimized first-order polynomial is used to roughly estimate the baseline contribution, and the offset and slope are added to the multiple line shapes as fitting parameters, thereby quantitatively obtaining the level of N-acetylneuraminyl glycoprotein from the NMR spectrum; preferably, the preprocessing of the NMR spectrum includes FID Fourier transform, phase adjustment, baseline correction, chemical shift calibration, and peak alignment; and 1D 1 The detection limit of H nuclear magnetic resonance spectrum is at μM level; preferably, the detection limit of 1D 1H nuclear magnetic resonance spectrum is 1 to 1000 μM; Preferably, the quality control measures of the nuclear magnetic resonance spectroscopy analysis include testing standard substances every day, testing mixed quality control (QC) biological samples prepared under the same conditions before testing each batch of biological samples, evaluating the reproducibility of the detection instrument based on the relative standard deviation (RSD) of the measurement results, and the reproducibility RSD of the detection instrument is <20%; adjusting the number of samples prepared daily according to the temperature equilibration time of the samples and the instrument tuning time to ensure that each sample obtains nuclear magnetic resonance spectroscopy data within 24 hours after preparation, thereby reducing biologically irrelevant deviations and interferences; before collecting the nuclear magnetic resonance spectroscopy spectrum, a deuterated methanol sample is used to calibrate the experimental temperature, a mixed standard substance reference sample is used for concentration quantitative calibration, and a sucrose reference sample is used for solvent peak suppression effect evaluation, thereby ensuring the stability of the detection environment and equipment; Preferably, the sample used for the nuclear magnetic resonance spectroscopy analysis is serum diluted with an equal volume of phosphate buffer, and the concentration of the phosphate buffer is 0.085M.

7. The use according to any one of claims 3 to 6, characterized in that: The system includes a data analysis module, which is used to calculate the Gensini score of the sample to be tested based on the level of N-acetylneuraminyl glycoprotein in the sample to be tested using a regression model, so as to evaluate the risk of coronary atherosclerotic heart disease or predict the risk of cardiovascular death in 5 years; preferably, the regression model is constructed and trained using the N-acetylneuraminyl glycoprotein test value and Gensini score of the known sample; further preferably, the regression model is a restricted cubic spline (RCS) regression model; further preferably, the regression model also includes a correction term, and the correction term is selected from age, gender, smoking, body mass index (BMI), systolic blood pressure (SBP) , fasting blood glucose (FPG), serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), brain natriuretic peptide (BNP), one or more of inflammatory markers; more preferably, a multivariate Logistics regression model is used to correct for age, gender, smoking, body mass index (BMI), systolic blood pressure (SBP), fasting blood glucose (FPG), serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), brain natriuretic peptide (BNP), one or more of inflammatory markers.

8. Use of reagents for detecting N-acetylneuraminyl glycoprotein in the preparation of kits for detecting the risk of coronary atherosclerotic heart disease or predicting the risk of cardiovascular death within 5 years; Preferably, the application targets adults; Or, the target of the application is Chinese; Alternatively, the sample to be tested is obtained from the body fluid of the subject of the application; more preferably, the sample to be tested is obtained from the blood or serum of the subject of the application.

9. The use according to claim 8, characterized in that: The reagent for detecting N-acetylneuraminyl glycoprotein is a reagent for detecting N-acetylneuraminic acid in N-acetylneuraminyl glycoprotein, or a reagent for detecting N-acetyl group in N-acetylneuraminyl glycoprotein.

10. The use according to any one of claims 3 to 9, characterized in that: The application is used in combination with other equipment, and the other equipment is selected from one or more of the following: coronary angiography, electrocardiogram, echocardiogram, exercise treadmill test, coronary CT angiography, radionuclide myocardial perfusion imaging, and cardiac magnetic resonance imaging.

11. Use of a reagent for detecting N-acetylneuraminyl glycoprotein in the preparation of a diagnostic agent, wherein: The diagnostic agent is used for diagnosing coronary atherosclerotic heart disease.

12. Use of a device or reagent for detecting N-acetylneuraminyl glycoprotein in a preparation system or kit, wherein: The system or kit is used for screening drugs for treating coronary atherosclerotic heart disease.

13. A device for detecting the risk of coronary atherosclerotic heart disease or predicting the risk of cardiovascular death within 5 years, characterized in that: The device comprises at least the following modules: N-acetylneuraminylglycine content detection module, used for detecting the content of N-acetylneuraminylglycine in the serum sample or blood sample of the test subject, detecting the N-acetylneuraminylglycine or N-acetyl group of N-acetylneuraminylglycine in the serum sample or blood sample of the test subject to determine the total content of N-acetylneuraminylglycine; A data analysis module is used to calculate the Gensini score of the sample to be tested based on the level of N-acetylneuraminyl glycoprotein in the sample to be tested using a regression model, so as to evaluate the risk of coronary atherosclerotic heart disease or predict the risk of cardiovascular death in 5 years; preferably, the regression model is constructed and trained using the N-acetylneuraminyl glycoprotein test value and Gensini score of the known sample; further preferably, the regression model is a restricted cubic spline (RCS) regression model; further preferably, the regression model also includes a correction term, and the correction term is selected from age, gender, smoking, body mass index (BMI), systolic blood pressure (SBP), fasting blood glucose (F More preferably, a multifactorial Logistics regression model is used to correct for age, gender, age, gender, smoking, body mass index (BMI), systolic blood pressure (SBP), fasting blood glucose (FPG), serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), brain natriuretic peptide (BNP), and one or more of inflammatory markers.

14. An electronic terminal, characterized in that: The electronic terminal includes: a processor, a memory, and a communicator; The memory is used to store a computer program; preferably, the computer program runs a restricted cubic spline (RCS) regression model and a multi-factor logistics regression model; The communicator is used to communicate with an external device to receive the detected N-acetylneuraminyl glycoprotein content data; The processor is used to execute the computer program stored in the memory so that the electronic terminal analyzes the N-acetylneuraminyl glycoprotein level after correcting the influence of the influencing factors to obtain the Gensini score of the sample to be tested, thereby evaluating the risk of coronary atherosclerotic heart disease or predicting the 5-year cardiovascular death risk.

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