Plasma biomarker of acute aortic dissection and its application

Abstract

FIELD: medicine.SUBSTANCE: use of a plasma biomarker for diagnosing acute aortic dissection (AAD), and a method for diagnosing AAD with such a marker. The use of isopentenyl adenine 7-N-glucoside as a diagnostic marker for the diagnosis of acute aortic dissection is proposed. A method for diagnosing acute aortic dissection in a subject is also provided, which comprises quantitatively determining isopentenyl adenine 7-N-glucoside in a whole blood sample of said subject, wherein an elevated level of isopentenyl adenine 7-N-glucoside compared to a healthy control subject indicates the presence of acute aortic dissection in said subject. The level of isopentenyl-adenine-7-N-glucoside in the blood of patients with AAD is significantly higher than in healthy individuals, which allows it to be used as a biomarker with high specificity and sensitivity for screening and diagnosis of acute aortic dissection.EFFECT: proposed diagnostic method is convenient and minimally invasive.4 cl, 1 dwg, 3 tbl, 1 ex

Description

[0001] Technical field

[0002] The present invention relates to the field of biomedicine, in particular to a specific plasma metabolic marker for acute aortic dissection and its use.

[0003] Technology Level

[0004] Acute aortic dissection (AAD) is the most dangerous aortic disease, the incidence of which is increasing worldwide. AAD is characterized by a pulsatile jet of blood entering the media through a rupture of the intima, causing dissection of the aortic wall. Despite improved treatment strategies, mortality and morbidity in patients with AAD remain high. According to inpatient records from the China Health Insurance Research Association (CHIRA) in 2011, the annual incidence of aortic dissection in China was estimated at 2.8 cases per 100,000 people, with approximately 48.6% of patients with aortic dissection dying before hospitalization. Furthermore, patients with aortic dissection in China tend to be younger than those in developed countries. There is growing evidence that aortic dissection is becoming a more severe disease in China. Furthermore, with OPA, clinical symptoms usually do not appear until an acute attack.Thus, early diagnosis and screening of OPA are of great importance for the prevention and treatment of this disease.

[0005] Current diagnostic methods for ORA primarily rely on imaging techniques, primarily chest CT. However, this detection method has a time lag and cannot provide a timely prediction of disease onset. Furthermore, some invasive radiographs cause additional harm to the body. It is generally believed that vascular structural and metabolic abnormalities are closely linked to the development of ORA. Metabolic phenotypes are highly sensitive to subtle variations and rapidly change in response to physiological and pathological stress. Therefore, analysis of low-molecular-weight metabolites in blood can clearly characterize the potential profile of a physiological system, thereby providing a rapid and reliable new approach to the early diagnosis of ORA.

[0006] According to previous studies, the levels of smooth muscle myosin heavy chain (smMHC), soluble human elastin fragment (sELAF), calponin, and soluble ST2 were significantly higher in patients with ORA than in healthy individuals. However, these various molecules, being proteins, are technically difficult to access, are expensive to analyze, and most have a limited half-life. Therefore, new biomarkers are needed for the early detection and prevention of ORA. The present invention provides a convenient, low-invasive, and inexpensive method for screening and diagnosing ORA using differential metabolic markers in the peripheral blood serum of a group of patients.

[0007] Essence of the invention

[0008] In view of the shortcomings of the prior art, the object of the present invention is to provide a new plasma biomarker for acute aortic dissection.

[0009] Another object of the present invention is to provide a plasma biomarker for acute aortic dissection.

[0010] Another object of the present invention is to provide a use of a reagent for determining a biomarker.

[0011] The objectives of the present invention can be realized through the following technical solutions:

[0012] Application of isopentenyl adenine-7-N-glucoside in the production of an auxiliary diagnostic reagent for acute aortic dissection. Isopentenyl adenine-7-N-glucoside is characterized by the structural formula shown below:

[0013] .

[0014] Application of isopentenyl adenine-7-N-glucoside detection reagent in preparing auxiliary diagnostic kit for acute aortic dissection.

[0015] In a preferred embodiment of the present invention, the reagent for detecting isopentenyl adenine-7-N-glucoside is a reagent for quantitatively detecting isopentenyl adenine-7-N-glucoside.

[0016] As another preferred embodiment of the present invention, a reagent for determining isopentenyl adenine 7-N-glucoside is a reagent for quantitatively determining isopentenyl adenine 7-N-glucoside using online solid phase extraction-liquid chromatography-tandem mass spectrometry.

[0017] Auxiliary diagnostic kit for acute aortic dissection contains:

[0018] 1) Plasma sample processing reagent;

[0019] 2) Isopentenyl adenine 7-N-glucoside quantitative determination reagent containing isopentenyl adenine 7-N-glucoside standard.

[0020] In a preferred embodiment of the present invention, the auxiliary diagnostic kit for acute aortic dissection comprises:

[0021] 1) Plasma sample processing reagent;

[0022] 2) Isopentenyl adenine-7-N-glucoside quantitative determination reagent using online solid phase extraction-liquid chromatography-tandem mass spectrometry, containing isopentenyl adenine-7-N-glucoside standard.

[0023] As another preferred embodiment of the present invention, a reagent for the quantitative determination of isopentenyl adenine-7-N-glucoside using online solid phase extraction-liquid chromatography-tandem mass spectrometry comprises:

[0024] a) Online solid phase extraction reagent comprising: an HLB SPE column with the following specifications: 5 μL and 20 mm × 3.9 mm; and mobile phase A: water, mobile phase B: methanol and mobile phase C: acetonitrile;

[0025] b) A reagent for liquid chromatography analysis, comprising: a Poroshell 120 EC-C18 chromatography column with the following specifications: 2.7 μL and 2.1 mm × 50 mm; and a mobile phase A: 10 mol / L ammonium acetate solution containing 0.1% formic acid, and a mobile phase B: acetonitrile containing 0.1% formic acid.

[0026] Preferred effects:

[0027] The present invention reveals that the metabolic molecule isopentenyl adenine-7-N-glucoside, which exhibits a significant difference in concentration between a patient with AAD and a healthy population, can be used as a biomarker with high specificity and sensitivity for screening and diagnosing acute aortic dissection. According to the application of the metabolic molecule, the metabolic molecule can be used to produce an auxiliary diagnostic reagent for AAD, which provides a convenient, low-invasive, and inexpensive method for screening and diagnosing subsequent aortic dissection and can provide an elective basis for an assessment system for accurate diagnosis and timely treatment evaluation of clinical patients.

[0028] Brief description of drawings

[0029] Fig. 1 shows the ROC curve of the results of assessing the risk of acute aortic dissection using the determination of isopentenyl-adenine-7-N-glucoside in plasma.

[0030] Detailed description of the invention

[0031] The technical solutions of the present invention are described below using specific examples. These examples are only some examples of the present invention and should not be construed as limiting the present invention. Various details in the examples may be appropriately changed without departing from the essence of the present invention.

[0032] Example 1

[0033] I. Experimental Materials and Methods

[0034] 1.1 Selection of subjects for clinical trial

[0035] The study subjects were enrolled from the Second Affiliated Hospital of Nanjing Medical University between January 2017 and April 2019. They were confirmed cases of acute aortic dissection according to the International Classification of Diseases, Tenth Revision, Clinical Modification of ICD-10 (ICD-10-CM) code I71 after obtaining written informed consent. Patients with Marfan syndrome, cancer, and connective tissue diseases were excluded. Healthy subjects in the control group were a healthy population who underwent a physical examination and routine blood tests during the same period. Patients with the disease and subjects in the control group were matched by age and gender and then identified. The study was approved by the ethics committee and was conducted in strict accordance with the ethical standards for human experimentation.

[0036] 1.2 Sample collection

[0037] Whole blood samples from patients were collected 72 hours after the attack, and blood samples from subjects in the control group were collected at the physical examination center. All anticoagulated blood samples were left at room temperature for 30 min and then centrifuged at 1000 g for 10 min to obtain plasma. Plasma samples were then quickly aliquoted and stored at -80°C. Before analysis, the samples were thawed on ice and then centrifuged for 15 min at 3000 rpm at room temperature, and the supernatant plasma was collected for analysis.

[0038] 1.3 Instruments and reagents

[0039] Methanol and Chloroform (CNW Technologies): HPLC Grade;

[0040] Acetonitrile (CAN, Fisher Chemical, USA): optimal purity for MS;

[0041] Ammonium acetate (purity ≥ 98%, Sigma-Aldrich, USA);

[0042] Ammonia aqueous solution (purity 28%-30%, Sigma-Aldrich, USA);

[0043] Isopentenyl adenine-7-N-glucoside (purity 99%, Sigma Aldrich Ltd);

[0044] clean water (from clean water filter);

[0045] Online solid phase extraction system 1260;

[0046] Liquid Chromatography 1260;

[0047] Triple Quadrupole Mass Spectrometer 6470;

[0048] Heraeus Fresco17 centrifuge (Thermo Fisher Scientific);

[0049] Forma 900 Series Ultra Low Temperature Freezer (Thermo Fisher Scientific);

[0050] ultrasonic device YM-080S;

[0051] DHG-9023A oven;

[0052] Vacuum dryer LNG-T98;

[0053] Electronic scale (Shanghai Weighing Instrument Factory);

[0054] Full-temperature oscillator (Changzhou Feipu).

[0055] 1.4 Sample Processing

[0056] A plasma sample (100 μL) was collected, chloroform:methanol (400 μL, 2:1 (v / v)) and the internal standard were added, mixed with rotation, and the mixture was shaken for 20 min. Then, the mixture was centrifuged at 13,000 rpm for 20 min at 4°C to separate the aqueous and organic layers. Then, the supernatant was transferred to a new tube and dried in a SpeedVac. After drying, the organic phase containing lipids was redissolved in chloroform:methanol (20 μL, 2:1 (v / v)) and isopropanol:acetonitrile:water (60 μL, 2:1:1 (v / v)). The solution was transferred to a 2 mL sample tube for instrument analysis. Equal volumes of samples (3 µL each) were collected, mixed, and used as a quality control (QC) sample. The QC sample was randomly assigned to the sample during detection and injected multiple times to determine the stability of the instrument and method.

[0057] 1.5 Detection and Analysis

[0058] The metabolite in each sample was analyzed using online solid-phase extraction-liquid chromatography-tandem mass spectrometry (XLC / MS / MS). The online solid-phase extraction system was connected to the liquid chromatographic separation system via a 10-way valve, and online enrichment and analysis of the samples were performed using the valve switching mode. For the online solid-phase extraction system, an HLB SPE column (5 μL and 20 mm × 3.9 mm, Waters Oasis) was used; mobile phase A was water, mobile phase B was methanol, and mobile phase C was acetonitrile. The specific gradient elution conditions are shown in Table 1. For the chromatographic system, a Poroshell 120 EC-C chromatographic column was used. 18(2.7 μL and 2.1 mm × 50 mm): where mobile phase A was 10 mol / L ammonium acetate solution (containing 0.1% formic acid) and mobile phase B was acetonitrile (containing 0.1% formic acid); the flow rate was 0.4 mL / min; the column temperature was 40°C; and the injection volume was 50 μL. The gradient elution conditions and the switching program of the 10-way valve are shown in Table 2.As for the mass spectrometer system, an electrospray ionization (ESI) source was used; the detection mode was the positive ion mode; the scanning mode was the dynamic multiple reaction monitoring (DMRM) mode; the capillary voltage was 3500 V, and the needle voltage was 500 V; the nitrogen temperature was 250°C, and the drying gas temperature was 300°C; the ammonia flow rate was 12 mL / min, and the drying gas flow rate was 6 mL / min; the nebulizer gas pressure was 241 kPa; the analysis time was 400 ms; the shortest residence time was 13.86 ms, and the longest residence time was 198.9 ms. The information on the corresponding gradient elution program is given in Table 1, and the information on the relevant parameters for mass spectrometry is given in Table 2.

[0059] Table 1 Specific parameters of the gradient elution program Time (min) Flow rate (ml / min) Mobile phase A (%) Mobile phase B (%) Mobile phase C (%) 0,00 0,3 100 0 0 4,00 0,3 100 0 0 4,01 0,3 0 100 0 8,00 0,3 0 100 0 8,01 2,0 0 0 100 11,00 2,0 0 0 100 11,01 3,0 100 0 0 12,00 0,3 100 0 0

[0060] Table 2 Switching program of 10-way valve Time (min) Mobile phase A (%) Mobile phase B (%) Valve connection 0,00 80 20 1 to 2 4,00 80 20 1 to 10 6,00 70 30 1 to 2 10,00 50 50 1 to 2 10,50 5 95 1 to 2 11,00 5 95 1 to 2 11,01 80 20 1 to 2 12,00 80 20 1 to 2

[0061] The mass spectrometer system was controlled and data collected using MassLynx v4.0 software, and the data were automatically processed by the QuanLynx application program. The tandem system was controlled using sparkLink v3.0 software.

[0062] II. Detection Results and Analysis

[0063] The analysis revealed the response values ​​of isopentenyl adenine-7-N-glucoside in the blood plasma of patients with aortic dissection and control subjects using the primary ESI+ and ESI- ionization modes. The metabolite profiles in the plasma of patients with acute aortic dissection significantly differed from those in the control group. Isopentenyl adenine-7-N-glucoside was significantly elevated in the patient group compared with the control group (Table 3). These results indicate that isopentenyl adenine-7-N-glucoside, as a metabolic marker, is associated with acute aortic dissection and can be used to predict acute aortic dissection. The ROC curve with the plasma isopentenyl adenine-7-N-glucoside level was constructed and analyzed. The results show AUC=0.9184 (Fig. 1), indicating a significant advantage for predicting acute aortic dissection.

[0064] Table 3. Expression levels of isopentenyl-adenine-7-N-glucoside in plasma of patients with aortic dissection and the control population, determined using mass spectrometry and P-test Control group A group of patients with acute aortic dissection P-test Isopentenyl adenine-7-N-glucoside 36,5±23,3 111,3±20,8 < 0,0001

[0065] The results indicate that isopentenyl adenine 7-N-glucoside in plasma can be used as a potential marker for the diagnosis of acute aortic dissection, with excellent diagnostic effect, which meets the requirements of convenience, low invasiveness and low cost, suitable for early screening of acute aortic dissection and suitable for auxiliary diagnosis and treatment evaluation.

Claims

1. Use of isopentenyl-adenine-7-N-glucoside as a diagnostic marker for the diagnosis of acute aortic dissection.

2. A method for diagnosing acute aortic dissection in a subject, comprising quantitatively determining isopentenyl adenine 7-N-glucoside in a whole blood sample of said subject, wherein an elevated level of isopentenyl adenine 7-N-glucoside compared to a healthy control subject indicates the presence of acute aortic dissection in said subject.

3. The method according to claim 2, wherein the quantitative determination of isopentenyl-adenine-7-N-glucoside is carried out using online solid-phase extraction-liquid chromatography-tandem mass spectrometry.

4. The method according to claim 3, wherein the quantitative determination of isopentenyl-adenine-7-N-glucoside using online solid-phase extraction-liquid chromatography-tandem mass spectrometry is carried out using: a) HLB SPE columns with the following characteristics: 5 µl and 20 mm × 3.9 mm; and mobile phase A: water, mobile phase B: methanol and mobile phase C: acetonitrile; b) a Poroshell 120 EC-C18 chromatographic column with the following characteristics: 2.7 µl and 2.1 mm × 50 mm; and mobile phase A: 10 mol / l ammonium acetate solution containing 0.1% formic acid, and mobile phase B: acetonitrile containing 0.1% formic acid.

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