WO2017161595A1 - Marker group for making diagnosis to distinguish coronary arterial atherosclerosis from stable angina pectoris - Google Patents

Abstract

A marker group for making diagnosis to distinguish coronary arterial atherosclerosis from stable angina pectoris. The marker group comprises one or more of glutathione, β-hydroxybutyric acid, proline, fumaric acid, phenylalanine and lactic acid. When a single marker is used for making diagnosis to distinguish patients with the stable angina pectoris from patients with the coronary arterial atherosclerosis, the areas under curve (AUC) of a ROC curve are all 0.7 or larger, and the marker has a clinical diagnosis significance; when the marker is used for diagnosis in combination, with the increase of the combination number, the AUC is further increased and reaches 0.988 when 6 markers are combined, and the sensitivity and the specificity are 98.5% and 98.3% respectively under the optimum cutoff value. The metabolic marker group can be used to accurately making diagnosis to distinguish the coronary arterial atherosclerosis from the stable angina pectoris, and has high sensitivity and specificity.

Description

Diagnosing a marker group that distinguishes between coronary atherosclerosis and stable angina Technical field

The invention belongs to the field of biochemistry and relates to a metabolic marker for diagnosis and classification of coronary heart disease, and particularly relates to a group of metabolic markers for diagnosis and differentiation of coronary atherosclerosis and stable angina pectoris.

Background technique

Coronary heart disease, also known as ischemic heart disease, involves atherosclerosis in the arteries supplying myocardial blood, that is, coronary atherosclerotic lesions cause vascular stenosis or plaque formation or even rupture, complete blockage, resulting in myocardial ischemia, hypoxia Or necrosis leads to ischemic heart disease, which causes a series of serious cardiovascular events such as clinical angina and myocardial infarction. Coronary heart disease is the main killer of human health. It has the characteristics of high incidence, high disability rate, high recurrence rate, high mortality rate and many complications. It has become a major disease threatening the health of our people.

At present, based on pathophysiological mechanisms, coronary heart disease is divided into chronic stable coronary heart disease (ie, stable angina) and acute coronary syndrome; acute coronary syndrome is further divided into unstable angina and acute myocardial infarction. Coronary heart disease from scratch, from light to heavy usually includes the following stages of development: normal coronary artery → coronary atherosclerosis → stable angina pectoris → unstable angina pectoris → acute myocardial infarction.

Coronary atherosclerosis is the main cause of coronary heart disease and is an early condition of coronary heart disease. Coronary atherosclerosis is a common progressive arterial disease. The lesions mainly involve moderately sized myometrial arteries, arterial intima lipid deposits, smooth muscle cell hyperplasia, and the formation of localized plaques that can harden the arterial wall. The rupture of the plaque leads to thrombosis, embolism, hemorrhage, partial or complete occlusion of the affected lumen, and clinical manifestations of atherosclerotic vascular complications. Early atherosclerotic lesions can occur before the age of 10 years. The disease causes arterial stenosis to take 20 to 30 years. It has no clinical symptoms in the early stage and is difficult to be discovered and valued. Therefore, it can be effective for early prevention and diagnosis of coronary atherosclerosis. To prevent the occurrence of coronary heart disease.

Coronary angiography can accurately determine the degree of coronary artery stenosis, and is the gold standard for the diagnosis of coronary heart disease [Comparative study of coronary angiography gold standard and clinical routine diagnosis of coronary heart disease, Shu Rongwen et al., Naval Medical Journal 2015 04] . As the gold standard for the diagnosis of coronary heart disease, coronary angiography can only find the degree of vascular stenosis, and it is also an interventional measurement method, requiring interventional surgery, and the diagnosis is expensive. On the other hand, the doctor also needs to make a final diagnosis based on the patient's electrocardiogram, cardiogram, treadmill exercise test, CT and other examination results. Due to the subjective judgment of the doctor and the unclear patient's narrative, the diagnosis of coronary heart disease still exists. Large misdiagnosis and missed diagnosis have a great impact on the prognosis of patients. In order to improve the quality of life of patients and reduce the life threats to patients, we urgently need to develop a diagnostic method with high diagnostic rate, economical, non-invasive and easy to operate.

Metabolomics is a science that studies the whole body of endogenous metabolites and their changes with internal and external factors. It is an important part of systems biology. It can perform rapid and non-invasive analysis of body fluids such as blood and urine. The difference in the spectrum gives a metabolic marker that can indicate various biochemical reactions. Currently commonly used analytical techniques include nuclear magnetic resonance (NMR), mass spectrometry (LC-MS/GC-MS), and the like. LC-MS/GC-MS has the characteristics of low sample preparation, high sensitivity, wide dynamic range, etc. It can be used to detect metabolites with large differences in concentration in samples, and thus become more and more applied in the study of metabolomics. platform. Plasma analysis is a commonly used disease diagnosis method in clinical practice and is widely used because of its advantages of simplicity, rapidity, economy, and relatively non-invasiveness.

Diagnostic typing of coronary heart disease has not been performed using plasma metabolite levels. The use of plasma metabolomics to find differences in plasma metabolite levels in patients with coronary arteries and coronary atherosclerosis and in patients with different types of coronary heart disease is important for rapid diagnosis and classification of coronary heart disease in early clinical stages.

Summary of the invention

In order to overcome the deficiencies of the prior art, a first object of the present invention is to provide a group of metabolic markers for diagnosing coronary atherosclerosis and stable angina pectoris, which are simultaneously present in plasma and can be simultaneously analyzed. Determination; a second object of the present invention is to provide a method capable of sensitively analyzing and detecting the metabolic marker population; and a third object of the present invention is to provide a detection kit based on the metabolic marker population, Diagnosis distinguishes between coronary atherosclerosis and stable angina, improves diagnostic convenience, and promotes standardization of diagnostic methods.

The above objectives are achieved by the following technical solutions:

A group of metabolic markers for the diagnosis of coronary atherosclerosis and stable angina, including one or more of the following metabolic markers: glutathione, beta-hydroxybutyrate, proline, rich Horse acid, phenylalanine, lactic acid.

Further, the metabolic marker population for diagnosing coronary atherosclerosis and stable angina comprises any two of said metabolic markers.

Further, the metabolic marker population for diagnosing coronary atherosclerosis and stable angina includes any of the three described metabolic markers.

Further, the population of metabolic markers for diagnosing coronary atherosclerosis and stable angina includes any of the four described metabolic markers.

Further, the metabolic marker population for diagnosing coronary atherosclerosis and stable angina comprises any of the five described metabolic markers.

Further, the metabolic marker population for diagnosing coronary atherosclerosis and stable angina comprises all six of said metabolic markers.

Further, the metabolic marker is a plasma metabolic marker.

A qualitative or quantitative analysis of the metabolic marker population for diagnosing coronary atherosclerosis and stable angina pectoris by using LC/MS and/or GC/MS for the metabolic markers Qualitative or quantitative analysis. The liquid and temperament detection limits are low and the sensitivity is high, and the metabolic markers in the biological samples can be sensitively analyzed and quantified.

A test kit for diagnosing coronary atherosclerosis and stable angina pectoris comprising a standard of said metabolic marker population, said standard being a chemical monomer or mixture of each metabolic marker. Standards can be used to quickly and accurately characterize and quantify metabolic markers in biological samples. The kit facilitates standardization of testing and improves ease of detection and reproducibility.

Further, the detection kit further comprises a solvent that dissolves the standard and/or a solvent that extracts the metabolic marker.

Advantages of the invention:

(1) The metabolic marker group provided by the present invention can accurately diagnose and distinguish between coronary atherosclerosis and stable angina pectoris. In the ROC curve evaluation method, the area under the ROC curve AUC is greater than 0.5, and the closer to 1, the better the diagnostic effect. AUC has lower accuracy from 0.5 to 0.7, AUC has a certain accuracy from 0.7 to 0.9, and AUC has higher accuracy at above 0.9. It has been verified that the AUC of the metabolic marker group provided by the present invention for diagnosis of coronary atherosclerosis and stable angina is above 0.7; in multiple combinations, the AUC is closer to 1 than the individual, diagnosis The effect is better; when the six joint applications, the AUC is closest to 1, the diagnosis is the best.

(2) The method for analyzing and detecting the metabolic marker provided by the present invention has high sensitivity and accurate and reliable results.

(3) The detection kit provided by the invention can be used for diagnosing coronary atherosclerosis and stable angina pectoris, improving the convenience of diagnosis, and promoting standardization of diagnostic methods.

detailed description

The substantial content of the present invention will be further described below in conjunction with the embodiments. The instruments or reagents used are not conventionally described as conventional instruments and reagents; the test methods not specifically described are conventional methods well known to those skilled in the art.

Example 1: Screening and Characterization of Plasma Differential Metabolites Between Patients with Coronary Atherosclerosis and Stable Angina Pectoris

I. Objects and methods

1. Specimen source

After obtaining the patient's consent, the peripheral blood plasma of 480 patients with coronary atherosclerosis, 280 patients with stable angina pectoris and 350 healthy people from September 2010 to June 2015 in Jiangsu Provincial People's Hospital were collected. All patients or health Per capita was confirmed by coronary angiography. The age and gender of healthy people matched with patients with coronary atherosclerosis and stable angina. All patients with coronary atherosclerosis, stable angina pectoris and healthy people have normal heart, lung, liver and kidney and hematopoietic function.

The blood collection time is in the early morning fasting state.

2, the main reagent

Acetonitrile and formic acid (UPLC pure) were purchased from ROE Company of the United States; chromatographic grade methanol and chloroform were purchased from Jiangsu Hanbang Technology Co., Ltd.; methoxyamine and N-methyl-N-(trimethylsilane)trifluoroacetamide (containing 1% trimethylchlorosilane) purchased from Sigma-Aldrich, USA; deionized water was prepared by Millipore's MIlli-Q ultrapure water system; The products include glutathione, β-hydroxybutyric acid, valine, fumaric acid, phenylalanine, and lactic acid, which were purchased from Sigma-Aldrich, USA.

3. Screening and characterization of plasma differential metabolites

3.1UPLC-Q/TOF-MS screening and characterization

3.1.1 Sample preparation

Extraction solvent optimization by response surface method: The extraction of rich metabolites in plasma by different solvents (acetonitrile, methanol, ethanol, chloroform, water) was investigated by the number of peaks and total peak area in mass spectrometry ESI+ and ESI-detection modes. Set efficiency. The experimentally measured data was subjected to multivariate analysis, and the importance of the variable to the model response was reflected by the importance importance of the PLS model. The acetonitrile, methanol, ethanol, chloroform and water have the highest VIP values of 1.503, 0.802, 0.651, 0.688 and 0.987. The extraction efficiency of acetonitrile is the highest, so acetonitrile is selected as the extraction solvent for plasma samples.

Sample processing: Take 100 μL of plasma in a 1.5 mL centrifuge tube, add 400 μL of acetonitrile, vortex for 30 seconds, mix, centrifuge at 13000 rpm × 10 min (4 ° C), take 200 μL of supernatant in a 1.5 mL centrifuge tube, and use nitrogen at room temperature. The mixture was blown dry, and the resulting residue was dissolved in 300 μL of a 20% aqueous acetonitrile solution.

3.1.2 Test conditions and parameters

UPLC-Q/TOF-MS conditions:

The chromatographic separation was performed by ultra performance liquid chromatography (UPLC, Agilent 1290, USA). The column was a Waters BEH C18 column (100 mm x 2.1 mm, 1.7 μm), the column temperature was 25 ° C, the injection chamber temperature was room temperature, and the injection volume was 2 μL. The positive and negative ion mode mobile phase compositions are all A with a volume concentration of 0.1% formic acid aqueous solution and B with a volume concentration of 0.1% formic acid acetonitrile solution. Gradient elution conditions: 0～1min is 0～30%B phase, B phase increases linearly to 60% in 2min, linearly changes to 90%B phase in 3～8min, then linearly increases to 100%B phase in 8～9min and then The temperature was maintained at 1 min; the flow rate was 0.3 mL/min, and the effluent after the column was directly introduced into the mass spectrometry system without splitting.

Mass spectrometry was performed using a quadrupole-time-of-flight mass spectrometer (Agilent 6530Q-TOF/MS, USA). Detected by electrospray ionization source (ESI) positive and negative ion mode; dry gas flow rate is 7L/min, dry gas temperature is 300 °C, dry gas and cone gas are high purity nitrogen; ion source temperature is 100 °C, positive ion and In negative ion mode, the capillary voltage is 3000V and the collision voltage is 100V. The data acquisition is performed three times per second in full scan mode. The scanning mass range is m/z 100-1000 Daltons.

3.2 GC-Q/MS screening characterization

3.2.1 Sample preparation

Take 200 μL of plasma in a 1.5 mL centrifuge tube, add 50 μL of 1 mg/mL 2-isopropylmalic acid solution to the internal standard, vortex for 20 seconds, and add 400 μL of a mixed solution of methanol, chloroform and water (2.5:1 ratio). :1), then shake on a metal bath at 70 ° C for 30 min (1200 rpm), centrifuge at 16000 g × 5 min (4 ° C), take 500 μL of the supernatant in a 1.5 mL centrifuge tube, add 500 μL of distilled water, vortex and mix, then 16000g After centrifugation (4 ° C) for 5 min, 500 μL of the supernatant was taken in a 1.5 mL centrifuge tube, and dried at room temperature with a nitrogen blower. The resulting residue was dissolved in 80 μL of methoxyamine pyridine solution at 50 ° C. After deuteration for 8 h, 60 μL of N-methyl-N-trimethylsilyltrifluoroacetamide was added and derivatized at 70 ° C for 2 h to obtain.

3.2.2 Test conditions and parameters

GC-Q/MS conditions: American Agilent 7890B-5977A gas chromatography-mass spectrometer. Column HP-5MS capillary column (30.0m × 0.25mm, capillary thickness 0.25μm); carrier gas is high purity helium, flow rate 1.0mL / min; injection volume 2μL; programmed temperature: 80 ° C constant temperature 2min, 80 ° C - 300 ° C (5 ° C / min) constant temperature 6 min; no split, injection temperature 300 ° C; interface temperature 300 ° C; ion source temperature 200 ° C; electron energy 50 eV; solvent delay 3 min; using full scan mode, scanning mass range: m / z 30-600 Daltons.

4, data processing and analysis

Data obtained from UPLC-Q/TOF-MS and GC-Q/MS were imported into SIMCA software (version 13.0.2, Umetrics) for multivariate statistical analysis. By establishing the OPLS-DA (orthogonal partial least squares-discriminant analysis) model, metabolites with high metabolic profiles (VIP>1.0 and p<0.01) were found between patients with coronary atherosclerosis and patients with stable angina.

Material structure retrieval by databases such as HMDB (http://www.hmdb.ca/) and Metline (http://metlin.scripps.edu/), using MS/MS obtained from accurate molecular weight and mass spectrometry provided in the database The map initially identified the structure of the above differential metabolites. The structure of the differential metabolites was finally confirmed by purchasing the standard, using the molecular weight of the standard, the chromatographic retention time, and the corresponding multi-stage MS fragmentation alignment.

Second, the results

Six differential metabolites were identified by screening: glutathione, β-hydroxybutyrate, valine, fumaric acid, phenylalanine, lactic acid.

Compared with patients with coronary arteriosclerosis, the above six differential metabolites were up-regulated or down-regulated in the plasma of patients with stable angina pectoris. By standard quantification, compared with patients with coronary arteriosclerosis, the expression level of the above differential metabolites in the plasma of patients with stable angina pectoris is: fumaric acid is down-regulated by 0.7-0.8 times; glutathione, β-hydroxybutyric acid, guanidine Amino acid, phenylalanine and lactic acid are respectively increased by 0.7 to 0.8 times. It can be seen that the above six differential metabolites have different expression levels in the plasma of patients with stable angina pectoris and coronary atherosclerosis, and can be used for diagnosis of coronary atherosclerosis and stable angina pectoris.

Example 2: Construction of ROC curves to verify the ability of six differential metabolites to diagnose coronary atherosclerosis and stable angina pectoris

The receiver operating curve (ROC) method was used to verify the differential expression levels of plasma metabolites in patients with stable angina and coronary atherosclerosis for the diagnosis of stable angina patients and patients with coronary atherosclerosis. Ability. The results showed that six differential metabolites of glutathione, β-hydroxybutyrate, proline, fumaric acid, phenylalanine and lactic acid were used alone to diagnose patients with stable angina and patients with coronary atherosclerosis. The ability is strong, the area under the ROC curve (AUC) is greater than 0.7, which has clinical diagnostic significance; when used in combination, the AUC is further improved with the increase of the number of joints, and the highest in all 6 joints, the AUC is 0.988. Sensitive at the best cutoff value The degree and specificity were 98.5% and 98.3%, respectively. The single and any 2 to 5 joint diagnosis results are shown in Tables 1-3.

Table 1 Single differential metabolite diagnosis distinguishes patients with coronary atherosclerosis and patients with stable angina

Single differential metabolite	AUC	Sensitivity	Specificity
Glutathione	0.878	87.7%	88.9%
--hydroxybutyric acid	0.865	84.4%	85.6%
Proline	0.843	82.2%	83.4%
Fumaric acid	0.806	78.5%	79.7%
Phenylalanine	0.790	76.9%	78.1%
Lactic acid	0.769	74.8%	76.0%

Table 2 Two differential metabolites combined diagnosis to distinguish patients with coronary atherosclerosis and stable angina pectoris

Table 3 any three to five differential metabolites combined diagnosis to distinguish patients with coronary atherosclerosis and stable angina pectoris

Joint number	AUC	Sensitivity	Specificity
Three	≥0.918	≥91.9%	≥91.3%
Four	≥0.930	≥92.4%	≥93.0%
Five	≥0.934	≥94.7%	≥94.5%

It can be seen from Table 1 that these six differential metabolites have a single ability to diagnose patients with coronary atherosclerosis and stable angina pectoris. The AUC is greater than 0.7, with high sensitivity and specificity. Diagnostic significance; it can be seen from Table 2 that when these six differential metabolites are used in combination for diagnosis, the AUC is higher than the single diagnosis, high sensitivity, high specificity, and has clinical diagnostic significance; It can be seen that when 3 to 5 of the 6 differential metabolites are used in combination for diagnosis, the AUC is further improved, the sensitivity is high, the specificity is strong, and the clinical diagnosis significance is obtained.

Therefore, these six differential metabolites can be used as metabolic markers for the diagnosis of coronary atherosclerosis and stable angina.

Example 3: Preparation of test kit

A detection kit is prepared based on the metabolic marker provided by the present invention, and the kit includes the following components:

Standards for metabolic markers: including glutathione, β-hydroxybutyric acid, proline, fumaric acid, phenylalanine, and lactic acid, each of which is packaged separately;

Plasma metabolite extraction solvent: 100% acetonitrile and 20% acetonitrile aqueous solution (for UPLC-Q/TOF-MS sample preparation); a ratio of 2.5:1:1 methanol, a mixed solution of chloroform and water, methoxyamine pyridine and N-methyl-N-trimethylsilyltrifluoroacetamide (for GC-Q/MS sample preparation); UPLC-Q/TOF-MS screening characterization, 20% aqueous acetonitrile can be used as a dissolution standard Solvent; GC-Q/MS screening characterization, using a plasma metabolite extraction solvent to prepare a standard solution by sample preparation;

Internal standard: 2-isopropyl malic acid.

Of course, when designing a test kit, a standard containing the above six metabolic markers is not required, and only a few of them can be used in combination. These standards can be packaged individually or in a mixture.

The kit is designed based on the metabolic markers provided by the present invention and can be used to diagnose patients with coronary atherosclerosis and stable angina.

In summary, the present invention effectively overcomes the deficiencies in the prior art and has a high industrial utilization value.

The above embodiments are intended to illustrate the substantial content of the present invention, but do not limit the scope of the present invention. A person skilled in the art should understand that the technical solutions of the present invention may be modified or equivalently substituted without departing from the spirit and scope of the technical solutions of the present invention.

Claims (10)

1. A group of metabolic markers for the diagnosis of coronary atherosclerosis and stable angina pectoris, characterized by comprising one or more metabolic markers as described below: glutathione, beta-hydroxybutyrate, guanidine Amino acid, fumaric acid, phenylalanine, lactic acid.
2. The metabolic marker population for diagnosing coronary atherosclerosis and stable angina pectoris according to claim 1, comprising any two of said metabolic markers.
3. The metabolic marker population for diagnosing coronary atherosclerosis and stable angina pectoris according to claim 1, comprising any three of said metabolic markers.
4. The metabolic marker population for diagnosing coronary atherosclerosis and stable angina pectoris according to claim 1, comprising any four of said metabolic markers.
5. The metabolic marker population for diagnosing coronary atherosclerosis and stable angina pectoris according to claim 1, comprising any five of said metabolic markers.
6. The metabolic marker population for diagnosing coronary atherosclerosis and stable angina pectoris according to claim 1, comprising all six of said metabolic markers.
7. The metabolic marker group for diagnosing coronary atherosclerosis and stable angina pectoris according to any one of claims 1 to 6, wherein the metabolic marker is a plasma metabolic marker.
8. A method for qualitatively or quantitatively analyzing a metabolic marker population for diagnosing coronary atherosclerosis and stable angina pectoris according to any one of claims 1 to 6, characterized in that: LC/MS and/or temperament are employed. The metabolic markers are qualitatively or quantitatively analyzed in combination.
9. A test kit for diagnosing coronary atherosclerosis and stable angina pectoris, comprising the standard of the metabolic marker group according to any one of claims 1 to 6, wherein the standard is each metabolism A chemical monomer or mixture of markers.
10. The test kit according to claim 9, further comprising a solvent that dissolves the standard and/or a solvent that extracts the metabolic marker.