Kit, biomarker and method

Abstract

The invention relates to the field of biomarkers, and discloses a kit, a biomarker and a method. The invention discloses a biomarker for early prediction and / or diagnosis of heart failure after myocardial infarction. The biomarker is short-chain fatty acid. The level of short-chain fatty acid in peripheral blood of a control group and an STEMI case group is quantitatively analyzed by using a high performance liquid chromatography-tandem mass spectrometer, and it is found that the content of the short-chain fatty acid in the STEMI case group is significantly reduced compared with the control group.

Description

Technical Field

[0001] This invention relates to the field of biomarkers, and more specifically to reagent kits, biomarkers, and methods. Background Technology

[0002] Heart failure (HF) is a common complication of myocardial infarction (MI). Currently, clinical assessment of HF risk after MI mainly relies on echocardiography (such as left ventricular ejection fraction measurement), serum biomarkers (such as B-type natriuretic peptide BNP and N-terminal pro-BNP), and cardiac magnetic resonance imaging (MRI). However, these methods have significant limitations. For example, echocardiography is affected by operator experience and is difficult to detect early compensatory changes in cardiac function; serum biomarkers (BNP / NT-proBNP) are specifically reduced in patients with renal insufficiency, and elevations often occur after HF symptoms appear, failing to provide early warning; while cardiac MRI can accurately assess myocardial fibrosis, it is expensive and not suitable for dynamic monitoring.

[0003] Current technologies do not address the discovery of biomarkers for pathological mechanisms such as myocardial energy metabolism disorders and gut-heart axis regulation, resulting in insufficient elucidation of the molecular mechanisms underlying HF progression. Currently, some studies have used non-targeted metabolomics to discover potential associations between short-chain fatty acids (SCFAs) such as acetic acid, propionic acid, and butyric acid and cardiovascular diseases (such as atherosclerosis and hypertension). However, the dynamic changes and mechanisms of SCFAs in HF after MI remain unclear, and no studies have publicly disclosed plasma SCFA levels as direct biomarkers for HF after MI.

[0004] Existing SCFA detection techniques mostly employ gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS), but these suffer from problems such as complex sample preparation (e.g., derivatization steps), low sensitivity (detection limit >10 nM), and the inability to simultaneously quantify multiple SCFAs. Furthermore, a clinical predictive tool based on the dynamic changes of SCFAs is lacking. Therefore, developing a novel SCFA detection technique is crucial for studying the dynamic changes of SCFAs in HF following MI. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing diagnostic methods for heart failure after myocardial infarction, which are complex and lack specificity.

[0006] To achieve the above objectives, a first aspect of the present invention provides a biomarker for early prediction and / or diagnosis of heart failure after myocardial infarction, said biomarker being a short-chain fatty acid.

[0007] A second aspect of the present invention provides a kit for diagnosing heart failure after myocardial infarction, the kit containing short-chain fatty acids from the biomarkers described in the first aspect as standards.

[0008] A third aspect of the present invention provides a method for determining the state of an individual using biomarkers, the method being used for non-disease diagnostic purposes, the method comprising:

[0009] (1) In the presence of internal standard and extractant, the test sample is extracted to obtain sample extract;

[0010] (2) The sample extract was analyzed by high performance liquid chromatography-tandem mass spectrometry to determine the content of the biomarker in the sample extract as C1; the biomarker is the biomarker described in the first aspect;

[0011] (3) Compare the content C1 of the biomarker determined in step (2) with the content C0 of the biomarker in the control group, and determine the status of the individual based on the comparison results obtained;

[0012] The control group consists of test samples from at least one group of individuals in the same condition;

[0013] The condition includes having heart failure after myocardial infarction and not having heart failure after myocardial infarction.

[0014] Through the above technical solution, the present invention has at least the following advantages over the prior art:

[0015] (1) This invention first discovered that SCFAs can serve as a novel biomarker for heart failure after myocardial infarction, breaking through the limitations of traditional methods for assessing heart failure after myocardial infarction. Moreover, changes in SCFAs levels occur 3-6 months earlier than heart failure symptoms, demonstrating significant clinical advantages and positive effects, enabling early warning of HF risk and providing a critical time window for early intervention.

[0016] (2) The present invention simplifies the sample pretreatment steps, eliminates plasma matrix interference, and improves detection efficiency by using solid phase microextraction technology to pretreat the sample.

[0017] (3) This invention optimizes high performance liquid chromatography-tandem mass spectrometry and combines it with solid phase microextraction technology to quantitatively analyze SCFAs with concentrations as low as 0.1 nM. The sensitivity is more than 100 times higher than that of traditional gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry. Moreover, it does not require complex derivatization steps, is easy to operate, and has controllable costs, making it suitable for large-scale clinical sample testing. Attached Figure Description

[0018] Figure 1 This is a graph showing the expression levels of propionic acid in the control group and the STEMI case group;

[0019] Figure 2 This is a graph showing the correlation analysis results between propionic acid and the degree of coronary artery stenosis after myocardial infarction;

[0020] Figure 3 This is a graph showing the survival rate curves of propionic acid and adverse cardiovascular events after myocardial infarction. Detailed Implementation

[0021] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0022] As previously stated, a first aspect of the present invention provides a biomarker for early prediction and / or diagnosis of heart failure after myocardial infarction, said biomarker being a short-chain fatty acid.

[0023] Preferably, the short-chain fatty acids include at least one selected from acetic acid, propionic acid, isobutyric acid, n-butyric acid, isovaleric acid, n-valeric acid, n-hexanoic acid, 3-hydroxyisovaleric acid, lactic acid, malonic acid, succinic acid, and glutaric acid. In this invention, by comparing the levels of SCFAs in the peripheral venous blood of individuals with ST-segment elevation myocardial infarction (STEMI) undergoing percutaneous coronary intervention (PCI) and control individuals, the inventors found that the concentration of SCFAs in the peripheral blood of STEMI patients was significantly lower than that of control individuals, and that SCFAs were negatively correlated with the severity of coronary artery stenosis. Therefore, short-chain fatty acids in peripheral venous blood are identified as microbial markers for heart failure after myocardial infarction.

[0024] As previously described, a second aspect of the present invention provides a kit for diagnosing heart failure after myocardial infarction, the kit containing short-chain fatty acids from the biomarkers described in the first aspect as standards.

[0025] As previously described, a third aspect of the present invention provides a method for determining the state of an individual using biomarkers, the method being used for non-disease diagnostic purposes, the method comprising:

[0026] (1) In the presence of internal standard and extractant, the test sample is extracted to obtain sample extract;

[0027] (2) The sample extract was analyzed by high performance liquid chromatography-tandem mass spectrometry to determine the content of the biomarker in the sample extract as C1; the biomarker is the biomarker described in the first aspect;

[0028] (3) Compare the content C1 of the biomarker determined in step (2) with the content C0 of the biomarker in the control group, and determine the status of the individual based on the comparison results obtained;

[0029] The control group consists of test samples from at least one group of individuals in the same condition;

[0030] The condition includes having heart failure after myocardial infarction and not having heart failure after myocardial infarction.

[0031] In a preferred embodiment, the method described in the third aspect of the present invention further includes: using a high-performance liquid chromatography-tandem mass spectrometer to quantitatively detect the standard to plot a concentration-response curve, converting the mass spectrometry signal into a concentration value, and monitoring the stability of each batch of experiments, thereby achieving accurate quantification and reliable analysis of SCFAs.

[0032] In a preferred embodiment, the standard is a pure standard of the target SCFAs.

[0033] According to a specific embodiment of the present invention, the method for preparing the standard is to dissolve 1 mg of acetic acid standard, 1 mg of propionic acid standard, 1 mg of isobutyric acid standard, 1 mg of n-butyric acid standard, 1 mg of isovaleric acid standard, 1 mg of n-valeric acid standard, 1 mg of n-hexanoic acid standard, 1 mg of 3-hydroxyisovaleric acid standard, 1 mg of lactic acid standard, 1 mg of malonic acid standard, 1 mg of succinic acid standard and 1 mg of glutaric acid standard in 1 mL of LC-MS grade ultrapure water.

[0034] Preferably, the test sample includes at least one of peripheral blood, feces, bone marrow, and urine.

[0035] Preferably, in step (1), the internal standard includes deuterated n-valeric acid and deuterated n-hexanoic acid; the extractant is acetonitrile. The inventors of this invention have found that in this preferred embodiment, the sample loading pretreatment steps for the chromatographic column are simplified, plasma matrix interference is eliminated, and detection efficiency is improved.

[0036] In this invention, the deuterated n-pentanoic acid refers to [2H9]-pentanoic acid.

[0037] In this invention, the deuterated hexanoic acid refers to [2H11]-hexanoic acid.

[0038] Preferably, in step (1), after the sample is extracted, 3-nitrophenylhydrazine solution and EDC-pyridine solution are added sequentially to perform chemical derivatization to obtain the sample extract.

[0039] Preferably, the volume ratio of the test sample, the 3-nitrophenylhydrazine solution, and the EDC-pyridine solution is 1.5-2.5:0.5-1.5:1.

[0040] According to a preferred embodiment, the volume ratio of the detection sample, the 3-nitrophenylhydrazine solution, and the EDC-pyridine solution is 2:1:1. The inventors of this invention have discovered that under this preferred condition, the concentration of target SCFAs in the detection sample can be accurately quantified, achieving ultra-high detection sensitivity while maintaining high operational efficiency.

[0041] Preferably, in step (2), the detection conditions of the high performance liquid chromatography-tandem mass spectrometry include: gradient elution, and the mobile phase of the liquid chromatography column is: solvent A: 0.1% (v / v)-0.2% (v / v) formic acid, solvent B: acetonitrile-methanol mixture; based on the total volume of the acetonitrile-methanol mixture, the volume ratio of acetonitrile to methanol is 1-3:1.

[0042] Preferably, the injection volume is 1 μL.

[0043] Preferably, in step (2), the flow rate of the mobile phase is 0.1-0.5 mL / min.

[0044] According to a preferred embodiment, the flow rate of the mobile phase is 0.35 mL / min.

[0045] Preferably, in step (2), the gradient elution conditions include: 0 min-2 min, A%:B% = 80%-60%: 20%-40%; 2-11 min, A%:B% = 30%-50%: 70%-50%; 11 min, A%:B% = 20%-65%: 80%-35%; 12 min, A%:B% = 60%-80%: 40%-20%, maintained for 13 minutes. In this preferred embodiment, % refers to volume percentage.

[0046] According to a preferred embodiment, the gradient elution conditions are as follows: A%:B% = 75%:25% for 0-2 min; A%:B% = 45%:55% for 2-11 min; A%:B% = 45%:55% for 11 min; A%:B% = 75%:25% for 12 min, and maintained for 13 minutes. In this preferred embodiment, % refers to volume percentage.

[0047] Preferably, in step (2), the detection conditions of the tandem mass spectrometer include: the curtain gas pressure is maintained at 35 psi, the collision energy is set to a moderate level (25 eV-35 eV) of collision-activated dissociation (CAD), the scanning mode is negative ionization mode, the spray voltage is set to -4500 V, the ion source temperature is adjusted to 450 °C, the pressure of the nebulizing gas (Gas1) is calibrated to 50 psi, and the pressure of the auxiliary heating gas (Gas2) is also adjusted to 50 psi. The inventors of this invention have found that under the above-mentioned preferred detection conditions of high-performance liquid chromatography-tandem mass spectrometry, this invention can achieve directional ionization enhancement, fragment-specific control, and matrix interference blocking, thereby enabling reliable detection of short-chain fatty acids in complex biological samples.

[0048] The present invention also includes a system for the early diagnosis of heart failure after myocardial infarction, the system comprising: a data storage device for storing expression level information of heart failure biomarkers after myocardial infarction; wherein the heart failure biomarkers after myocardial infarction are the biomarkers described in the first aspect; a data analysis device for calculating the risk of heart failure after myocardial infarction based on the expression level information of heart failure biomarkers after myocardial infarction; and a result output device for outputting the results of the data analysis device.

[0049] Preferably, a significant decrease in the biomarker indicates a high risk of heart failure following myocardial infarction.

[0050] The present invention will be described in detail below through examples. Unless otherwise specified, the methods used in the following examples are conventional methods in the art, and the reagents used are all commercially available. Room temperature refers to 25±2℃.

[0051] Preparation method of 50% acetonitrile aqueous solution: Dissolve 50 mL of acetonitrile in 50 mL of deionized water.

[0052] Preparation method of 6% pyridine solution: Dissolve 6 mL of pyridine in 94 mL of 50% acetonitrile aqueous solution.

[0053] The method for preparing 3-nitrophenylhydrazine solution is as follows: Dissolve 1.224 mg of 3-nitrophenylhydrazine in a 50% (v / v) aqueous solution of acetonitrile.

[0054] The EDC-pyridine solution is prepared by dissolving 0.920 mg EDC and 2.4 μL of pyridine solution in 37.6 μL of 50% acetonitrile aqueous solution.

[0055] The AB Sciex / Waters ultra-high performance liquid chromatography-mass spectrometry system (AB Sciex Qtrap5500 / Waters I-class) was used.

[0056] [2H9]-valeric acid was purchased from Zhenzhun Company, catalog number IR32270.

[0057] [2H11]-hexanoic acid was purchased from Zhenzhun Company, product number IR-30252.

[0058] Acetonitrile was purchased from Fisher Scientific, product number A998-4.

[0059] Pyridine was purchased from Aladdin, catalog number P141169-1L.

[0060] 3-Nitrophenylhydrazine was purchased from Sigma, catalog number N21804-25g.

[0061] Example 1

[0062] This embodiment is used to illustrate the grouping of the control group and the STEMI case group, and the medical record information of the STEMI case group;

[0063] The STEMI case group included 50 adult patients aged 19-87 years who were diagnosed with ST-segment elevation myocardial infarction (STEMI) and underwent percutaneous coronary intervention (PCI) at Beijing Tongren Hospital, affiliated with Capital Medical University, between April and December 2023.

[0064] The inclusion criteria for ST-segment elevation myocardial infarction are: ischemic chest discomfort plus ≥1 of the following: electrocardiographic changes (ST-segment elevation ≥ elevation ≥ electrocardiogram) and elevated cardiac markers.

[0065] Exclusion criteria for STEMI cases included the following conditions at admission: cardiogenic shock; sepsis; active malignancy; or end-stage renal disease (defined as estimated glomerular filtration rate [eGFR] <15 mL / min / 1.73 mcg). 2 Anemia: hemoglobin <8g / dL for men and <7g / dL for women; taking antibiotics before or during hospitalization; inflammatory bowel disease; inflammatory vascular disease.

[0066] The control group included 50 patients aged 19-87 years who underwent routine health screening at Beijing Tongren Hospital, affiliated with Capital Medical University, between April and December 2023, and who had no prior diagnosis of acute myocardial infarction (AMI). The control group also met the above exclusion criteria.

[0067] All direct percutaneous coronary intervention (PCI) procedures are performed by board-certified interventional cardiologists who perform more than 500 PCI procedures annually.

[0068] In this invention, 'door-to-balloon time' is defined as the time from admission (designated "door") to the start of PCI procedure (designated "balloon"). Most STEMI patients seek medical attention within 12 hours of the onset of myocardial infarction symptoms and undergo percutaneous coronary intervention within 24 hours.

[0069] Peripheral venous blood was collected from all enrolled control group patients. Prior to PCI surgery, peripheral venous blood was systematically collected from all enrolled STEMI cases.

[0070] The patient's medical record information mainly includes: the angiography results of direct percutaneous coronary intervention (PPCI) for STEMI patients, clinical information, and patient follow-up information; among which, clinical information includes demographic characteristics, blood and urine biochemical indicators, echocardiography, etc.

[0071] The Gensini score was used to assess STEMI patients; the scoring criteria are detailed in the reference (DOI:10.20039 / j.cnki.1007-3949.2025.06.008). The SYNTAX score was calculated using an open digital platform and the validated SYNTAX scoring method (http: / / www.syntaxscore.com).

[0072] Comprehensive demographic data, clinical characteristics, and treatment strategies for both the control and STEMI case groups were systematically retrieved from electronic health records. Outcome assessment combined retrospective graphical analysis with prospective follow-up interactions with patients or their designated agents.

[0073] The primary endpoints include major adverse cardiovascular events (MACE), particularly cardiovascular mortality, HF-related readmissions, and recurrent vascular events (myocardial infarction and stroke). The operational definition of cardiovascular death is sudden death of unknown cause or death due to acute myocardial infarction (AMI), decompensated heart failure, or life-threatening arrhythmias. HF readmission criteria require a hospital stay ≥24 hours, documented worsening of HF symptoms, and appropriate adjustment of HF-related pharmacological treatment.

[0074] Post-admission monitoring showed that the median follow-up period was 3 months, and 48 participants (follow-up rate of 96%) completed follow-up of adverse cardiovascular events.

[0075] Example 2

[0076] In this embodiment, targeted metabolomics was used to identify the structure and determine the concentration of SCFAs in peripheral venous blood of control group and STEMI patients.

[0077] The detection was performed using an ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS / MS) system. Detailed operating procedures are as follows:

[0078] (1) Peripheral venous blood samples from the control group and STEMI case group collected in Example 1 were centrifuged at 3000 rpm for 10 minutes at 4°C to separate plasma from cells. The clear plasma layer was aliquoted into cryovials and stored at -80°C for the determination of SCFAs concentration.

[0079] (2) The following standardized procedure is adopted for sample pretreatment:

[0080] 200 μL of acetonitrile solution containing deuterated internal standard, pre-cooled at 4 °C, was added to 100 μL of plasma to obtain a mixture. The mixture was vigorously vortexed for 60 seconds, followed by ultrasonic extraction under ice-water bath conditions for 10 minutes. The mixture was then centrifuged at 12000 rpm for 10 minutes at 4 °C to achieve phase separation. 80 μL of the supernatant was transferred to an analytical vial. The acetonitrile solution containing deuterated internal standard refers to acetonitrile containing [2H9]-valeric acid and [2H11]-hexanoic acid. The solution is prepared by adding 2 μL of 1 mM [2H9]-valeric acid and 2 μL of 1 mM [2H11]-hexanoic acid to 200 μL of acetonitrile.

[0081] (3) The derivatization steps for samples and standards are as follows:

[0082] Add 40 μL of 200 mM 3-nitrophenylhydrazine solution and 40 μL of 120 mM EDC-pyridine solution sequentially to 80 μL of supernatant or 80 μL of standard solution. Activate at 40 °C for 30 min to ensure complete derivatization, then immediately quench on ice for 60 s, and freeze at -80 °C for LC-MS / MS analysis. The standard solutions were prepared by dissolving 1 mg of acetic acid standard, 1 mg of propionic acid standard, 1 mg of isobutyric acid standard, 1 mg of n-butyric acid standard, 1 mg of isovaleric acid standard, 1 mg of n-valeric acid standard, 1 mg of n-hexanoic acid standard, 1 mg of 3-hydroxyisovaleric acid standard, 1 mg of lactic acid standard, 1 mg of malonic acid standard, 1 mg of succinic acid standard, and 1 mg of glutaric acid standard in 1 mL of LC-MS grade ultrapure water.

[0083] Key experimental parameters include: 1. Acetonitrile solution containing deuterated internal standard, 3-nitrophenylhydrazine solution and EDC-pyridine solution were all stored at -20℃ before use; 2. Quality control samples (QC) were prepared from extracts of peripheral venous blood samples.

[0084] (4) Chromatographic conditions: The column was an ACQUITY UPLC BEH C18 analytical column (100×2.1mm, 1.7μm); the injection volume was 1μL; the mobile phase flow rate was 0.35mL / min; the column temperature was 40℃; the mobile phase was: solvent A: 0.1% (v / v) formic acid, solvent B: acetonitrile-methanol mixture; the elution gradient was: 0-2min, 75% (v / v)A / 25% (v / v)B; 2-11min, 45% (v / v)A / 55% (v / v)B; 11min, 45% (v / v) / 55% (v / v)%; at 12min, reequilibrate to the baseline conditions (75% (v / v)A / 25% (v / v)B) and maintain for 13min. The preparation method for 0.1% (v / v) formic acid is as follows: 0.1 mL of formic acid is added to 99.9 mL of LC-MS grade ultrapure water; the acetonitrile-methanol mixture contains acetonitrile and methanol in a volume ratio of 2:1.

[0085] (5) Mass spectrometry operating parameters include: curtain pressure maintained at 35 psi, collision energy set to a medium level of collision activated dissociation (CAD), negative ionization mode, spray voltage set to -4500V, ion source temperature adjusted to 450℃, nebulizing gas (Gas1) pressure calibrated to 50 psi, and auxiliary heating gas (Gas2) pressure also adjusted to 50 psi.

[0086] (6) The theoretical framework for compound identification and quantification operates through the following mechanism: Quantitative analysis of metabolites is performed using a multiple reaction monitoring (MRM) method implemented on a triple quadrupole mass spectrometer. In this detection paradigm, a primary quadrupole separates specific precursor ions (precursor ions) associated with the target analyte, effectively eliminating isotopic interferences through mass-to-charge ratio filtration. These separated precursor ions then undergo controlled fragmentation in a collision chamber via collision-induced dissociation, generating diagnostic fragment ions. A tertiary quadrupole then selectively transports unique fragment ions specific to the target molecule, thus establishing a two-stage mass filtration that effectively eliminates non-specific ion species, optimizes measurement accuracy, and improves experimental repeatability. Post-acquisition data processing includes comprehensive peak integration of all sample datasets and systematic normalization of peak areas of the same chemical entities detected in different biological matrices to maintain analytical comparability.

[0087] Example 3

[0088] This embodiment illustrates the Logrank test survival analysis and Pearson correlation analysis performed on the measured SCFAs and the medical record information of the STEMI case group, including: correlation analysis of SCFAs with the degree of coronary artery stenosis (i.e., correlation with SYNTAX and Gensini) and correlation analysis of SCFAs with MACEs events in STEMI patients.

[0089] First, an independent samples t-test was used to assess the difference in SCFA expression levels between the control group and the STEMI case group. Results are as follows: Figure 1 As shown. From Figure 1 The results showed that the peripheral blood SCFAs level in the STEMI case group was significantly lower than that in the control group.

[0090] Pearson correlation analysis was used to analyze the relationship between propionic acid and the severity of coronary artery stenosis after myocardial infarction (SYNTAX score). Pearson correlation analysis was also used to analyze the relationship between propionic acid and the severity of coronary artery stenosis after myocardial infarction (Gensini score). The results of the correlation analysis between propionic acid and the severity of coronary artery stenosis after myocardial infarction are as follows: Figure 2 As shown. From Figure 2 As can be seen from the data, propionic acid is negatively correlated with the severity of coronary stenosis after myocardial infarction.

[0091] Logrank test survival analysis was used to analyze the prognostic effects of propionic acid on cardiovascular adverse events following myocardial infarction. Cardiovascular adverse events included recurrent myocardial infarction, stroke, readmission due to heart failure, and cardiac death. The prognostic analysis results are as follows: Figure 3 As shown. Logrank test survival analysis was used to analyze the prognostic effects of propionic acid on cardiovascular events following myocardial infarction. Cardiovascular events included myocardial infarction, stroke, and cardiac death. The prognostic analysis results are shown below. Figure 3 As shown. From Figure 3 The study revealed that the lower the concentration of propionic acid in the peripheral blood of STEMI patients, the worse the prognosis after myocardial infarction. This clarifies the correlation between peripheral blood propionic acid levels and heart failure after myocardial infarction.

[0092] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A biomarker for early prediction and / or diagnosis of heart failure after myocardial infarction, characterized in that, The biomarker is a short-chain fatty acid.

2. The biomarker according to claim 1, characterized in that, The short-chain fatty acids include at least one of acetic acid, propionic acid, isobutyric acid, n-butyric acid, isovaleric acid, n-valeric acid, n-hexanoic acid, 3-hydroxyisovaleric acid, lactic acid, malonic acid, succinic acid, and glutaric acid.

3. A reagent kit for diagnosing heart failure after myocardial infarction, characterized in that, The kit contains short-chain fatty acids from the biomarkers described in claim 1 or 2 as standards.

4. A method for determining the state of an individual using biomarkers, the method being used for non-disease diagnostic purposes, characterized in that, The method includes: (1) In the presence of internal standard and extractant, the test sample is extracted to obtain sample extract; (2) The sample extract was analyzed by high performance liquid chromatography-tandem mass spectrometry to determine the content of the biomarker in the sample extract as C1; the biomarker is the biomarker described in claim 1 or 2; (3) Compare the content C1 of the biomarker determined in step (2) with the content C0 of the biomarker in the control group, and determine the status of the individual based on the comparison results obtained; The control group consists of test samples from at least one group of individuals in the same condition; The condition includes having heart failure after myocardial infarction and not having heart failure after myocardial infarction.

5. The method according to claim 4, characterized in that, The test samples include at least one of peripheral blood, feces, bone marrow, and urine.

6. The method according to claim 4, characterized in that, In step (1), the internal standard includes deuterated n-valeric acid and deuterated n-hexanoic acid; the extractant is acetonitrile.

7. The method according to claim 4, characterized in that, In step (1), after the sample is extracted, 3-nitrophenylhydrazine solution and EDC-pyridine solution are added sequentially to perform chemical derivatization to obtain the sample extract. Preferably, the volume ratio of the test sample, the 3-nitrophenylhydrazine solution, and the EDC-pyridine solution is 1.5-2.5:0.5-1.5:

1.

8. The method according to claim 4, characterized in that, In step (2), the detection conditions of the high performance liquid chromatography-tandem mass spectrometry include: gradient elution, and the mobile phase of the liquid chromatography column is: solvent A: 0.1% (v / v)-0.2% (v / v) formic acid, solvent B: acetonitrile-methanol mixture; based on the total volume of the acetonitrile-methanol mixture, the volume ratio of acetonitrile to methanol is 1-3:1; Preferably, the injection volume is 1 μL.

9. The method according to claim 4, characterized in that, In step (2), the flow rate of the mobile phase is 0.1-0.5 mL / min.

10. The method according to claim 4, characterized in that, In step (2), the gradient elution conditions include: 0 min-2 min, A%:B% = 80%-60%: 20%-40%; 2-11 min, A%:B% = 30%-50%: 70%-50%; 11 min, A%:B% = 20%-65%: 80%-35%; 12 min, A%:B% = 60%-80%: 40%-20%, maintained for 13 minutes.

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