Application of biotinidase in the preparation of reagents for the detection of heart failure with preserved ejection fraction in diabetic patients

Abstract

The present invention relates to the field of biotechnology, and in particular to the use of biotinidase in the preparation of reagents for detecting heart failure with preserved ejection fraction in diabetic patients. The present invention has found through research that biotinidase has high sensitivity and high specificity for predicting the occurrence of heart failure with preserved ejection fraction in diabetic patients, and preliminary cell experiments have shown that biotinidase has a significant protective effect on diabetes combined with heart failure with preserved ejection fraction. Therefore, the present invention applies biotinidase to the preparation of reagents for detecting heart failure with preserved ejection fraction in diabetic patients. When the reagent is used for the detection of heart failure with preserved ejection fraction in diabetic patients, it is simple to operate, can simplify the detection process, and help reduce the workload of medical staff; and the detection time is shortened and the cost is low; at the same time, it can also predict multiple subjects at one time, and can be used for large-scale population screening.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of biotinidase in preparing a reagent for detecting heart failure with preserved ejection fraction in diabetic patients. Background Art

[0002] Heart failure is the terminal stage of cardiovascular disease and poses a serious threat to human health. Among all types of heart failure, heart failure with preserved ejection fraction (HFpEF) is a syndrome with complex pathogenic factors, often accompanied by metabolic comorbidities and diastolic dysfunction. In recent years, HFpEF has accounted for more than 50% of the total number of heart failure patients, and the number of patients is still growing rapidly at a rate of 1% per year, making HFpEF a major problem in the field of heart failure. Due to the complex pathogenic mechanism of HFpEF, traditional anti-heart failure drugs, except for sodium-glucose cotransporter 2 inhibitors, have not obtained large-scale evidence-based medicine. Therefore, actively searching for the key mechanisms and factors that regulate HFpEF is of great significance to improving HFpEF and finding new therapeutic targets.

[0003] Diabetes mellitus (DM) is one of the important causes of HFpEF and has a significant negative impact on its prognosis. The impact of DM on cardiovascular mortality and heart failure readmission rate in HFpEF patients is higher than that in patients with heart failure with reduced ejection fraction (HFrEF) (HR 2.0 vs. HR1.6). The proportion of heart failure hospitalization in HFpEF patients with DM is 2.5 times that of non-DM patients. HFpEF patients with DM have more obvious cardiac ultrasound abnormalities, more signs of congestion, higher NT-proBNP levels, and worse quality of life and prognosis. It can be seen that DM is closely related to a significant increase in the incidence of complications in HFpEF patients during and after discharge, but the pathophysiological mechanism of DM-induced HFpEF is still unclear.

[0004] Currently, according to the ACC Expert Consensus Decision Path for the Management of Heart Failure with Preserved Ejection Fraction (HFpEF) published in 2023, the H2FPEF scoring system and the HFA-PEFF scoring system are the most reliable criteria for diagnosing HFpEF. However, the H2FPEF scoring system and the HFA-PEFF scoring system are designed with complex test methods (including echocardiography, electrocardiogram, cardiac magnetic resonance imaging, and even cardiac tissue biopsy, etc.), a single test takes a long time, the test cost is high, and the amount of information required is high, which is not conducive to follow-up work and large-scale population screening and prediction work.

[0005] Therefore, the prior art still needs to be improved and developed. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide the use of biotinidase in the preparation of a reagent for detecting heart failure with preserved ejection fraction in diabetic patients, aiming to solve the problems of complex detection process, long time and high cost of existing detection methods.

[0007] The technical solution of the present invention is as follows:

[0008] The present invention provides application of biotinidase in preparing a reagent for detecting heart failure with preserved ejection fraction in diabetic patients.

[0009] Optionally, the reagent comprises a biotinidase standard.

[0010] Optionally, the reagent comprises a biotinidase antibody.

[0011] Optionally, the biotinidase antibody is labeled with a fluorescent tag.

[0012] Optionally, the reagent further comprises a horseradish peroxidase-labeled secondary antibody and a color developer.

[0013] Optionally, the developer is 3,3',5,5'-tetramethylbenzidine.

[0014] Optionally, the reagent is used for the detection of heart failure with preserved ejection fraction in diabetic patients, comprising:

[0015] Collect whole blood from the subject to be tested, add anticoagulant, and obtain an initial sample;

[0016] Pre-treating the initial sample to obtain a serum sample;

[0017] detecting the concentration of biotinidase in the serum sample by using the reagent;

[0018] The concentration of the biotinidase is used as a detection index to predict whether diabetic patients will develop heart failure with preserved ejection fraction.

[0019] Optionally, the anticoagulant is selected from one or more of ethylenediaminetetraacetic acid, sodium citrate, sodium citrate, and sodium heparin.

[0020] Optionally, the pretreatment is centrifugation or polymer material filtration.

[0021] Optionally, the prediction standard is: when the concentration of the biotinidase is between 0-2.204 nmol / L, it is predicted that heart failure with preserved ejection fraction will occur; when the concentration of the biotinidase is greater than 2.204 nmol / L, it is predicted that heart failure with preserved ejection fraction will not occur.

[0022] Beneficial effects: The present invention applies biotinidase to the preparation of a reagent for detecting heart failure with preserved ejection fraction in diabetic patients. When the reagent is used for detecting heart failure with preserved ejection fraction in diabetic patients, compared with the prior art, the advantages are: simple operation, simplified detection process, and helpful in reducing the workload of medical staff; shortened detection time and low cost; multiple subjects can be predicted at one time, and it has the potential to be used for large-scale population screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a diagram of the differential protein sequencing results of human plasma samples in Example 1.

[0024] Figure 2 This is a diagram of the differential pathway sequencing results of human plasma samples in Example 1.

[0025] Figure 3 This is a diagram of the differential protein sequencing results of the cell samples in Example 1.

[0026] Figure 4 This is a diagram of the sequencing results of differential pathways of cell samples in Example 1.

[0027] Figure 5 This is a graph of BTD content in the sequencing results of human plasma samples in Example 1.

[0028] Figure 6 This is a graph showing the experimental results of the effect of knocking down BTD on cardiomyocytes and their mitochondria in Example 2.

[0029] Figure 7 This is a graph showing the experimental results of the effect of exogenous BTD supplementation on cardiac structure and function and myocardial mitochondria in Example 2.

[0030] Figure 8 This is a flow chart for predicting the occurrence of HFpEF in diabetic patients by detecting BTD levels in Example 3.

[0031] Fig. 9 This is a graph comparing the effectiveness of detecting BTD levels, H2FPEF and HFA-PEFF in predicting the occurrence of HFpEF in diabetic patients in Example 3. DETAILED DESCRIPTION

[0032] The present invention provides the use of biotinidase in the preparation of a reagent for the detection of heart failure with preserved ejection fraction in diabetic patients. In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0033] The energy metabolism process of cardiomyocytes is affected by the circulatory microenvironment in which they are located. When the blood components are abnormal, pathological changes in their structure and function may occur. In order to find the mechanism of energy metabolism reconstruction of cardiomyocytes when DM and HFpEF are combined, the present invention incubates the human cardiomyocyte cell line (PromoCell, C-12810) with the blood of DM and DM combined with HFpEF people in DMEM culture medium, thereby simulating the blood microenvironment under the conditions of DM and DM combined with HFpEF. Metabolomics detection was performed 48 hours later, and it was found that the metabolic process of the two groups of cardiomyocytes was significantly different ( Figure 1 ), KEGG analysis revealed that the differential metabolites were mainly enriched in the "vitamin B metabolism" pathway ( Figure 2 In order to further search for pathogenic substances in the blood, the present invention conducted proteomics testing on the blood of the two groups of people and found that the protein profiles in the blood of the two groups of people changed significantly ( Figure 3 ). KEGG analysis revealed that the differentially expressed proteins were mainly enriched in the “vitamin digestion and absorption” pathway ( Figure 4 ), and among the proteins enriched in this pathway, the expression level of biotinidase (BTD) was significantly decreased ( Figure 5 ). Therefore, DM may be the main mechanism that promotes the occurrence of HFpEF by causing a decrease in circulating BTD levels.

[0034] BTD is widely present in mammals and is an important coenzyme that promotes the separation of biotin from binding proteins and the formation of free biotin. Biotin, also known as vitamin B8 or vitamin H, is a water-soluble sulfur-containing vitamin. Like other B vitamins, most of it is taken in from food, and a few are synthesized in bacteria in the intestines of the body. As a cofactor of biotin-dependent enzymes (biotin-dependent carboxylase, decarboxylase and transcarboxylase), biotin is linked to lysine residues through covalent bonds and participates in metabolic processes such as fatty acid synthesis, amino acid metabolism and gluconeogenesis. In addition, BTD can also mediate the binding of biotin groups to lysine sites on histones H2, H3, and H4, resulting in histone biotinylation modification. Biotinylation plays a role in cell proliferation, DNA repair and maintaining genome stability.

[0035] When BTD is deficient, the intestinal ability to absorb biotin is reduced, the cleavage and recycling of biotin bound to proteins in the body is reduced, leading to biotin deficiency in the body, thereby reducing the activity of biotin-dependent carboxylases, affecting the catabolism of sugars, fats and amino acids, and manifesting as metabolic disorders and poisoning symptoms. In addition, BTD and biotin deficiency can cause a decrease in the level of histone biotinylation in the transposon region, increase the activity of the transposon, and thus lead to genomic instability. Blood BTD levels are an important basis for diagnosing BTD deficiency. The present invention found that the blood BTD and biotin levels were lower in patients with DM and HFpEF; in the primary cardiomyocyte model, knocking down BTD can significantly promote cardiomyocyte hypertrophy, activate oxidative stress and affect the mitochondrial oxidative respiration process, while supplementing BTD can significantly improve this phenomenon. In order to further explore the role of BTD in HFpEF caused by DM, the present invention administered BTD synthetic protein to DM mice by tail vein injection, and the results showed that exogenous supplementation of BTD can significantly improve cardiomyocyte hypertrophy, cardiac diastolic dysfunction, and mitochondrial structural and functional abnormalities caused by DM. The above results indicate that BTD deficiency can cause cardiac energy metabolism disorders, and exogenous supplementation of BTD has a protective function on the heart.

[0036] Based on the above research results, an embodiment of the present invention provides the use of biotinidase in preparing a reagent for detecting heart failure with preserved ejection fraction in diabetic patients.

[0037] Specifically, the present invention has found through screening and verification that there are significant differences in the levels of biotinidase in the DM population and the DM combined with HFpEF population (i.e., HFpEF-5 subtype), and the sensitivity and specificity of biotinidase for predicting the occurrence of HFpEF in DM patients are significantly higher than those of other HFpEF subtypes, and preliminary cell experiments have shown that biotinidase has a significant protective effect on DM combined with HFpEF. Therefore, biotinidase can be used to prepare reagents for the detection of heart failure with preserved ejection fraction in diabetic patients.

[0038] In one embodiment, the reagent includes a biotinidase standard substance, and the biotinidase standard substance is used to establish a standard curve of biotinidase concentration, thereby determining the concentration of biotinidase in the sample to be tested.

[0039] In one embodiment, the reagent comprises a biotinidase antibody, which can recognize biotinidase in the sample to be tested.

[0040] In one embodiment, the biotinidase antibody carries a fluorescent label. When the biotinidase antibody carries a fluorescent label, the concentration of the biotinidase can be determined by detecting the fluorescence intensity.

[0041] In one embodiment, the reagent further comprises a horseradish peroxidase labeled secondary antibody and a color developer. When the biotinidase antibody does not carry a fluorescent label, it can be combined with a horseradish peroxidase (HRP) labeled secondary antibody, and then a color developer is added to react with HRP to develop color, and the concentration of the biotinidase is determined by detecting the absorbance.

[0042] In one embodiment, the color developing agent is 3,3',5,5'-tetramethylbenzidine. 3,3',5,5'-tetramethylbenzidine (TMB) is a common substrate of HRP, but is not limited thereto and can be replaced by other substances that can react with HRP to develop color.

[0043] In one embodiment, the reagent is used for the detection of heart failure with preserved ejection fraction in diabetic patients, comprising:

[0044] Collect whole blood from the subject to be tested, add anticoagulant, and obtain an initial sample;

[0045] Pre-treating the initial sample to obtain a serum sample;

[0046] detecting the concentration of biotinidase in the serum sample by using the reagent;

[0047] The concentration of the biotinidase is used as a detection index to predict whether diabetic patients will develop heart failure with preserved ejection fraction.

[0048] In one embodiment, the anticoagulant is selected from one or more of ethylenediaminetetraacetic acid, sodium citrate, sodium citrate, and heparin sodium.

[0049] In one embodiment, the pretreatment is centrifugation or filtration of polymer materials.

[0050] In one embodiment, the detection method is enzyme-linked immunosorbent assay or liquid chromatography-mass spectrometry.

[0051] In one embodiment, the prediction standard is: when the concentration of the biotinidase is between 0-2.204 nmol / L, it is predicted that heart failure with preserved ejection fraction will occur; when the concentration of the biotinidase is greater than 2.204 nmol / L, it is predicted that heart failure with preserved ejection fraction will not occur.

[0052] The present invention will be further described below by means of specific examples.

[0053] Example 1

[0054] This example screened biomarkers for heart failure with preserved ejection fraction in diabetic patients, as follows:

[0055] (1) Data Collection

[0056] A total of 20 patients with diabetes who visited the University of Hong Kong-Shenzhen Hospital between January 1, 2020 and December 31, 2022 were collected, including 10 patients with simple DM and the other 10 patients with DM and HFpEF.

[0057] Simple DM patients were defined as those with HbA1c ≥ 6.5% (or random blood glucose ≥ 11.1 mmol / L; or fasting blood glucose ≥ 7.0 mmol / L; or 2-hour blood glucose after an oral glucose tolerance test ≥ 11.1 mmol / L), and without congenital heart structural defects or other combined diseases.

[0058] Definition of DM combined with HFpEF patients: ① Patients have symptoms of heart failure: dyspnea, limited activity and fluid retention; ② Left ventricular ejection fraction ≥ 50%, no left ventricular enlargement; ③ Patients have structural heart disease (such as left atrial enlargement, left ventricular hypertrophy) and / or diastolic dysfunction. If E / e'>15, then heart failure; if 8<E / e'<15, and one of the following conditions, such as NT.proBNP>125pg / mL, E / A<1, left atrial enlargement, left ventricular hypertrophy, and atrial fibrillation, can also be determined as diastolic dysfunction. The above three items must be met at the same time to be determined as DM combined with HFpEF.

[0059] Exclusion criteria: ① Patients with valvular heart disease, pericardial disease, hypertrophic cardiomyopathy, and restrictive cardiomyopathy as indicated by cardiac ultrasound; ② Acute myocardial infarction, tuberculosis, chronic obstructive pulmonary disease, liver and kidney failure, tumors, major trauma, surgery, infection, blood disease, immune disease, and other diseases that may cause changes in biomarker levels within the past month.

[0060] Blood collection and pretreatment method: whole blood was collected from the median cubital vein, anticoagulated with EDTA, centrifuged at 3000 rpm for 10 min, and the top layer of serum was taken for later use.

[0061] Cell collection and pretreatment method: Human cardiomyocyte cell line was cultured in DMEM medium containing plasma from patients with DM or DM combined with HFpEF for 24 hours, the medium was discarded, the medium was washed with PBS, the liquid was discarded, PBS was added, and the cells were gently scraped with a cell scraper and collected into EP tubes, and centrifuged at 500 rpm for 5 minutes.

[0062] (2) Proteome sequencing process of cells or serum samples

[0063] ① Enzymatic hydrolysis

[0064] Take an equal amount of each sample protein for enzymatic hydrolysis, add an appropriate amount of standard protein, adjust the volume to the same with lysis buffer, and then add dithiothreitol (DTT) to a final concentration of 5mM, and reduce at 56°C for 30min. Then add iodoacetamide (IAA) to a final concentration of 11mM, and incubate at room temperature in the dark for 15min. Transfer the alkylated sample to an ultrafiltration tube, centrifuge at 12000g for 20min at room temperature, replace with 8M urea 3 times, and then replace urea with replacement buffer 3 times, add trypsin at a ratio of 1:50 (protease: protein, m / m), and enzymatic hydrolysis overnight. Centrifuge at 12000g for 10min at room temperature to recover the peptides, and then use ultrapure water to recover the peptides once, and combine the two peptide solutions.

[0065] ② LC-MS scanning analysis

[0066] The peptides were dissolved in liquid chromatography mobile phase A and separated using the NanoElute ultra-high performance liquid phase system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an acetonitrile-water solution containing 0.1% formic acid. The liquid phase gradient was set as follows: 0-10min, 9%-24% B; 10.0-14.0min, 24%-35% B; 14.0-17.0min, 35%-80% B; 17.0-20.0min, 80% B, and the flow rate was maintained at 1000nl / min. After separation by the ultra-high performance liquid phase system, the peptides were injected into the Capillary ion source for ionization and then into the timsTOF Pro (Bruker) mass spectrometer for data acquisition. The ion source voltage was set to 2.0kV, and the peptide parent ions and their secondary fragments were detected and analyzed using TOF. The data acquisition mode used was data-independent parallel accumulation serial fragmentation (dia-PASEF) mode. The primary mass spectrometry scanning range was set to 100-1700 m / z. After one primary mass spectrum was acquired, 10 PASEF mode acquisitions were performed. The secondary mass spectrometry scan was in the range of 100-1700 m / z, with every 25 m / z as a window.

[0067] ③Search database

[0068] Database retrieval is a complex calculation process, which requires the use of professional mass spectrometry data analysis software for data analysis. The raw data from the mass spectrometer is imported into the search software, and the corresponding analysis parameters are set according to the experimental plan. The secondary mass spectrometry data of this experiment was retrieved using Proteome Discoverer (v2.4.1.15). Retrieval parameter settings: The database is Homo_sapiens_9606_PR_20210721.fasta (78120 sequences), and the anti-library is added to calculate the false positive rate (FDR) caused by random matching; the enzyme cutting method is set to Trypsin (Full); the number of missed cutting sites is set to 2; the minimum length of the peptide is set to 6 amino acid residues; the maximum number of peptide modifications is set to 3; the mass error tolerance of the primary parent ion is set to 10ppm, and the mass error tolerance of the secondary fragment ion is 0.02Da. Carbamidomethyl (C) is set as a fixed modification, and Oxidation (M), Acetyl (N-terminus), Met-loss (M), and Met-loss + acetyl (M) are set as variable modifications. The FDR for protein, peptide, and PSM identification was set to 1%.

[0069] ④ Notes

[0070] Gene Ontology (GO), protein domain, KEGG pathway, COG / KOG functional classification and subcellular localization were selected for protein annotation.

[0071] ⑤Quantitative analysis

[0072] The search results give the LFQ intensity of each protein in different samples (the result of the original intensity value of the protein after correction between samples). The LFQ intensity (I) of the protein in different samples is transformed by centering to obtain the relative quantitative value (R) of the protein in different samples. The calculation formula is as follows:

[0073] Rij = Iij / Mean (Ij);

[0074] Where i represents sample and j represents protein.

[0075] ⑥ Differential protein screening

[0076] Three or more replicates: First, select the samples to be compared, and take the ratio of the mean relative quantitative values ​​of each protein in multiple replicate samples as the fold change (FC). For example, calculate the fold change of proteins between sample group A and sample group B. The calculation formula is as follows:

[0077] FCA / B,k=Mean(Rik,i∈A) / Mean(Rik,i∈B);

[0078] Where R represents the relative quantitative value of protein, i represents sample, and k represents protein.

[0079] In order to determine the significance of the difference, the relative quantitative value of each protein in the comparison group samples was subjected to a T test, and the corresponding P value was calculated as a significance indicator, with the default P value < 0.05. In order to make the test data conform to the normal distribution required by the T test. Before the test, the relative quantitative value of the protein needs to be transformed by Log2. The calculation formula is as follows:

[0080] Pk=T.test(Log2(Rik,i∈A),Log2(Rik,i∈B)).

[0081] Repeat twice: First, select the samples to be compared, and take the mean of the relative quantitative values ​​of each protein in the two repeated comparison groups as the fold change (FC). For example, calculate the fold change of the protein between sample group A and sample group B. The calculation formula is as follows:

[0082] FCA / B,k=Mean((Rik,i∈A) / (Rik,i∈B));

[0083] Where R represents the relative quantitative value of protein, i represents sample, and k represents protein.

[0084] In order to determine the significance of the difference, the standard coefficient of variation (CV) of each protein in the two comparison groups was calculated as a significance indicator, and the default CV<0.1. The calculation formula is as follows:

[0085] CVk=SD(A1k / B1k,A2k / B2k) / Mean(A1k / B1k,A2k / B2k).

[0086] No duplication: First, select the samples to be compared, and take the ratio of the relative quantitative values ​​of each protein in the sample as the fold change (FC). For example, calculate the fold change of the protein between sample A and sample B. The calculation formula is as follows:

[0087] FCA / B,k=RAk / RBk;

[0088] Where R represents the relative quantitative value of protein, and k represents protein.

[0089] Through the above difference analysis, when P value < 0.05, the difference expression change exceeding 1.5 was regarded as the threshold of significant upregulation, and less than 1 / 1.5 was regarded as the threshold of significant downregulation.

[0090] ⑦Create a volcano map

[0091] Through the above differential analysis, when P value < 0.05, the difference expression change of more than 1.5 was used as the threshold of significant upregulation, and less than 1 / 1.5 was used as the threshold of significant downregulation. The differential protein volcano map was established with log2FC as the horizontal axis and -log10P value as the vertical axis.

[0092] (3) Sequencing results

[0093] The sequencing results are as follows Figure 1-5 As shown. Among them, Figure 1 Volcano map of metabolomics sequencing of human cardiomyocytes after incubation of plasma from DM and DM combined with HFpEF for 48 hours, green: down-regulated; red: up-regulated, n = 6; Figure 2 KEGG analysis for metabolomics sequencing, red box: Vitamin B metabolism; Figure 3 Volcano plot of plasma proteomic sequencing in DM and DM combined with HFpEF population, green: down-regulated; red: up-regulated, n = 10; Figure 4 KEGG analysis of proteomics sequencing, red box: Vitamindigestion and absorption; Figure 5 Proteomic sequencing analysis of BTD expression levels in the two groups, n = 10, **P < 0.01. DM: diabetes; DM + HFpEF, diabetes combined with heart failure with preserved ejection fraction.

[0094] Depend on Figure 1-5 It can be seen that, ① Cell sequencing results: The metabolic processes of the two groups of cardiomyocytes were significantly different. KEGG analysis found that the differential metabolites were mainly enriched in the "vitamin B metabolism" pathway. It is very similar to the "vitamin digestion and absorption" pathway in the serum protein group, and BTD ( Figure 1-2 ). ② Plasma sequencing results: The protein profiles in the blood of the two groups of people changed significantly. KEGG analysis results showed that the differentially expressed proteins were mainly enriched in the "vitamin digestion and absorption" pathway ( Figure 4 ), and among the proteins enriched in this pathway, the BTD of the two groups of serum samples was significantly different, as shown by a significant decrease in expression level ( Figure 3 , Figure 5 ). Therefore, DM may be the main mechanism that promotes the occurrence of HFpEF by causing a decrease in circulating BTD levels.

[0095] Example 2

[0096] This example further explores the role of BTD in HFpEF caused by DM, as follows:

[0097] (1) Effects of BTD knockdown on cardiomyocytes and their mitochondria

[0098] ①Cell transfection and drug treatment

[0099] Three groups were set up: control group (si-NC), BTD knockdown group (si-BTD), and BTD knockdown + BTD recombinant protein group (si-BTD + RecBTD). Among them, cells in the si-NC group were transfected with negative control small interfering RNA for 48 hours, cells in the si-BTD group were transfected with BTD small interfering RNA for 48 hours, and cells in the si-BTD + Rec BTD group were transfected with BTD small interfering RNA for 48 hours. At 24 hours, BTD recombinant protein was added to make the final concentration reach 1nmol / L.

[0100] ②Western blot detection

[0101] Protein lysis: Treat the cell samples with RIPA lysis buffer containing 1% PMSF, lyse on ice for 30 min, centrifuge at 12000 g for 15 min, and transfer the supernatant to a new EP tube.

[0102] Pre-sampling treatment: Quantification, add corresponding PBS and 5×SDS Loading buffer to the protein sample, boil in boiling water for 5 minutes, and prepare for loading after instant separation.

[0103] SDS-PAGE electrophoresis and electrotransfer: electrophoresis was first performed at 70 V for 30 min, then at 120 V for 60 min; electrotransfer was performed at a constant current of 230 mA for 100-120 min.

[0104] Blocking: Block with 5% skim milk for 1 hour, wash with TBST for 5 minutes each time, for a total of three times.

[0105] Primary antibody incubation: The strips were incubated with ANF, BNP, β-MHC, CI-NDUFB8, CII-SDHB, CIII-UQCRC2, and CV-ATP5A antibodies at 4°C overnight.

[0106] Secondary antibody incubation: Incubate with secondary antibody at 1:5000 for 1 hour.

[0107] Development: The strips were incubated with the mixed luminescent solution for 2 min and then developed in a BioRad developer.

[0108] Grayscale calculation: Image J was used for grayscale analysis, and the grayscale ratio of the target band to the internal reference protein band was used to compare the expression differences among the three treatment groups of si-NC, si-BTD, and si-BTD+Rec BTD.

[0109] ③ATP detection

[0110] Prepare samples: lyse cells or tissues with protein lysis buffer, centrifuge at 10,000 g for 5 min at 4°C, and carefully remove the supernatant into a new EP tube; add Reagent A and mix well, centrifuge at 10,000 g for 5 min, and discard the supernatant; add pre-cooled Reagent B; incubate at 4°C for 30 min, vortexing for 5 seconds every 5 minutes; centrifuge at 16,000 g for 20 min, and remove the supernatant into a new pre-cooled 1.5 ml centrifuge tube for subsequent operations.

[0111] Add reagents: Set up the fluorescence spectrophotometer or fluorescence microplate reader (temperature 37°C, excitation wavelength 530nm, emission wavelength 590nm, gain multiple 100); prepare 5 1.5 ml centrifuge tubes, marked as tubes 1 to 5, and add different volumes of buffer Reagent C to tubes 1-5 according to the gradient; transfer 20μl of standard solution (Reagent F) to tubes 1-5 and mix; place on ice and avoid light. Standard sample determination Transfer 20μl Reagent C to a new cuvette, add Reagent F, Reagent D and Reagent E, pour up and down several times, mix, and incubate at 37°C for 30min;

[0112] Immediately measure using a fluorescence spectrophotometer: obtain relative fluorescence readings;

[0113] Construct a standard curve: the vertical axis (Y axis) is the relative fluorescence unit; the horizontal axis (X axis) is the standard ATP concentration (μmol / L). Sample measurement Pipette Reagent C into a new cuvette, add 50μl of the sample to be tested, add Reagent D and Reagent E, pour up and down several times, mix, incubate at 37℃ for 30min, and immediately put it into the fluorescence spectrophotometer for detection: obtain the relative fluorescence reading, and calculate the corresponding ATP concentration (μmol / L) of the sample according to the standard curve.

[0114] ④ROS detection

[0115] Slice embedding: Take cardiomyocyte slices and embed them directly into the compound with the optimal cutting temperature;

[0116] DHE incubation: Superoxide production in the heart was performed using dihydroethylene (DHE) in a humidified chamber at 37°C in the dark for 45 min;

[0117] Analysis: Image-Pro Plus software was then used to analyze the mean fluorescence intensity of each group of cells.

[0118] ⑤Test results

[0119] The results of the test on the effect of knocking down BTD on cardiomyocytes and their mitochondria are as follows Figure 6 As shown. Among them, A is the expression level of ANF and BNP related proteins in each group detected by Western blot, n=5; B is the expression level of mitochondrial complex proteins in each group detected by Western blot, n=5; C is the ATP synthesis level of cardiomyocytes in each group, n=5; D is the ROS production of cardiomyocyte mitochondria, the picture is magnified 200 times, scale: 200μm. Compared with the si-NC group, *P<0.05, **P<0.01, ***P<0.001, p value less than 0.05 indicates that the data of the study group are significantly different from the data of the si-NC group, and the smaller the p value data, the greater the probability of a significant difference between the two groups; compared with the si-BTD group, #P<0.05, p value less than 0.05 indicates that the data of the study group are significantly different from the data of the si-BTD group, and the smaller the p value data, the greater the probability of a significant difference between the two groups; Rec BTD: BTD recombinant protein.

[0120] Depend on Figure 6 It can be seen that compared with the si-NC group, the expression levels of hypertrophy markers ANF, BNP, and β-MHC in myocardial cells in the si-BTD group were significantly increased, the contents of electron transport chain-related proteins CI-NDUFB8, CII-SDHB, CIII-UQCRC2, CV-ATP5A and ATP were decreased, and the level of ROS was significantly increased. Compared with the si-BTD group, the expression levels of ANF, BNP, and β-MHC in the si-BTD+Rec BTD group were significantly decreased, and the contents of electron transport chain-related proteins CI-NDUFB8, CII-SDHB, CIII-UQCRC2, CV-ATP5A and ATP were significantly increased. This suggests that BTD improves myocardial hypertrophy effects at the cellular level, promotes the expression of electron transport chain-related proteins and ATP generation, and reduces ROS generation.

[0121] (2) Effects of exogenous BTD supplementation on cardiac structure and function and myocardial mitochondria

[0122] ① Mouse grouping and setting

[0123] Three groups were set up, including the control group (Control), the model group (DM), and the model + BTD group (DM + Rec BTD). Among them, the control group was fed with ordinary feed for 24 weeks; the DM group was fed with a high-fat diet containing 60% fat for 12 consecutive weeks. In the 4th week, 35 mg / kg of streptozotocin was injected intraperitoneally to induce hyperglycemia; the mice in the model + BTD group were fed with a high-fat diet containing 60% fat for 24 consecutive weeks. In the 4th week, 35 mg / kg of streptozotocin was injected intraperitoneally once to induce hyperglycemia. Starting from the 21st week, 10 μg / mouse of IGF1 recombinant protein was injected into the tail vein once a week for 4 consecutive times until the end of the 24th week.

[0124] ②WGA staining

[0125] After the experiment, the mice were anesthetized with isoflurane for echocardiography, and then killed by intraperitoneal injection of an overdose of tribromoethanol anesthetic, and the heart was removed. The heart was evenly divided into two parts along the anatomical horizontal plane, and the upper part was used for WGA staining. The specific steps are:

[0126] Slicing: The blade should be sharp and thin, the tissue thickness should be about 2-3mm, and the size should be 1.5×1.5×0.2-0.3cm.

[0127] Fixation: After the tissue is removed, it should be immediately placed in 10% formalin (equivalent to 4% formaldehyde) for fixation.

[0128] Rinse: Rinse in running water for 2-10 hours.

[0129] Dehydration: From low concentration of alcohol to high concentration of alcohol, the gradient is as follows: 70% (5-10min) → 80% (60-120min) → 90% (60-120min) → 95% (60-120min) → 95% (60-120min) → 100% (60-120min).

[0130] Staining: Place the slices in PBS and add WGA fluorescent marker, incubate at room temperature for 2 hours; then wash the slices in PBS for 3 times, 5 minutes each time; then add DAPI fluorescent marker to the slices and incubate at room temperature for 30 minutes, and finally wash the slices in PBS for 3 times, 5 minutes each time.

[0131] Photography: The images were taken under a Zeiss fluorescence microscope at a magnification of 200 times.

[0132] ③ Mouse heart ultrasound

[0133] The equipment used was Vevo 2100 echocardiography system, and the modes included standard imaging plane, M-mode, color mode, Doppler and functional calculation; observation mode setting and parameter acquisition: the mice underwent ultrasound under isoflurane anesthesia, and the parasternal left ventricular short-axis view was used to measure EF, FS, LVID, GLS, LV volume, EDV, and SV; from the top four-chamber view, the left ventricular pressure-volume loop (PV loop), E / A, E / e', and E peak deceleration time change values ​​were measured under Doppler, and the heart rate (HR) was recorded at the same time.

[0134] ④ Transmission electron microscopy

[0135] Sampling: Remove a small piece of tissue from the heart, so that the tissue piece is less than 1mm 3 .

[0136] Fixation: Fix with 2.5% glutaraldehyde in phosphate buffer for 2 hours, rinse with 0.1M phosphoric acid solution for 15 minutes each time, for a total of three times; fix with 1% osmium acid fixative for 2-3 hours.

[0137] Rinse: Rinse three times with 0.1 M phosphoric acid solution, 15 min each time.

[0138] Dehydration: Treat with ethanol-acetone mixtures of different proportions at 4°C, as follows: 50% ethanol, 15-20 min; 70% ethanol, 15-20 min; 90% ethanol, 15-20 min; 90% ethanol-90% acetone (1:1), 15-20 min; 90% acetone, 15-20 min. Finally, dehydrate with pure acetone at room temperature three times, 5 min each time.

[0139] Embedding: Treat the tissue with different gradients of acetone + embedding solution. The specific gradients are as follows: a. Pure acetone + embedding solution (2:1) at room temperature for 3-4 hours; b. Pure acetone + embedding solution (1:2) at room temperature overnight; c. Pure embedding solution at 37°C for 2-3 hours.

[0140] Curing: Use a temperature gradient to cure the embedded tissue, as follows: a. 37°C oven overnight; b. 45°C oven for 12 hours; c. 60°C oven for 24 hours.

[0141] Sectioning: Ultrathin sectioning, thickness 50-60nm.

[0142] Staining: 3% uranyl acetate-lead citrate double staining.

[0143] Photography on the machine: observation and photography under transmission electron microscope.

[0144] ⑤ATP detection

[0145] The same specific implementation process as (1) in this embodiment ③: ATP detection.

[0146] ⑥Test results

[0147] The results of the test on the effect of exogenous BTD supplementation on cardiac structure and function and myocardial mitochondria are as follows Figure 7 As shown. A is the representative WGA staining image and statistical graph of ventricular muscle in each group, with a magnification of 200 times, scale bar = 50μm, n = 5; B is the change of E / A value of cardiac ultrasound in each group of mice, n = 10; C is the change of E peak deceleration time of cardiac ultrasound in each group of mice, n = 10; D is the electron microscope scanning image of ventricular tissue in each group of mice, with a magnification of 15000 times, scale bar: 2μm; E is the change of ATP synthesis of isolated cardiomyocytes in cardiac tissue of each group of mice, n = 5, *P < 0.05, **P < 0.01, ***P < 0.001. Control: control group; DM: diabetes combined with HFpEF group; DM + Rec BTD: diabetes combined with HFpEF + exogenous supplementation of BTD recombinant protein group.

[0148] Depend on Figure 7 As shown in Figure A, the surface area of ​​cardiomyocytes in the DM group mice increased compared with that in the Control group. Figure 7 As shown in Figures B and C, the diastolic function of the mice in the DM group was impaired, including a decrease in the E / A value and a prolonged E peak deceleration time. Figure 7 As shown in Figure D, the myofilaments of the ventricular muscles of the normal group mice were closely arranged, and the mitochondria were mostly rod-shaped or club-shaped. In the DM group, the myofilaments were loosely arranged, the number of mitochondria increased, the mitochondria were obviously swollen, and some mitochondria changed from long rods to nearly round shapes. Figure 7 As shown in Figure E, the ATP synthesis capacity of myocardial cells in the DM group was significantly lower than that in the control group. Compared with the DM group, the above abnormal indicators in the DM+Rec BTD group were significantly improved. The above results show that, at the in vivo level, exogenous supplementation of BTD can improve myocardial hypertrophy, cardiac diastolic dysfunction, and mitochondrial structural and functional abnormalities caused by DM.

[0149] In summary, this example shows that BTD deficiency can cause cardiac energy metabolism disorders, and exogenous supplementation of BTD has a protective function on the heart.

[0150] Example 3

[0151] In this example, the occurrence of HFpEF in diabetic patients is predicted by detecting the BTD level. The flow chart is as follows: Figure 8 and compared it with the prediction performance of H2FPEF and HFA-PEFF.

[0152] (1) Screening of user population

[0153] Inclusion criteria: age ≥18 years, HbA1c ≥6.5% (or random blood glucose ≥11.1mmol / L; or fasting blood glucose ≥7.0mmol / L; or 2-hour blood glucose after oral glucose tolerance test ≥11.1mmol / L), and no congenital heart structural defects.

[0154] Exclusion criteria: age <18 years or HbA1c <6.5% or congenital heart disease.

[0155] (2) Blood sample pretreatment

[0156] Blood sample collection: Whole blood was collected from the median cubital vein of the subjects using blood collection tubes. To prevent blood clotting, blood was collected using blood collection tubes containing a 1:10 anticoagulant such as sodium citrate or EDTA.

[0157] Blood sample pretreatment: Place the test tube containing blood into a centrifuge and centrifuge at 3000rpm for 10min. The blood sample is separated into three layers. Take the top layer as the blood component and put it into a new EP tube.

[0158] (3) Sample testing steps

[0159] ① Coating enzyme-labeled reaction 8-tube strip: In an 8*12 polystyrene flat-bottom columnar 8-tube strip, dilute the BTD antibody at 1:1000 with pH 9.6 NaHCO3 buffer, add 100 μl of the diluted BTD antibody solution to each well, incubate at 4°C overnight, discard the antibody solution, wash with NaHCO3 buffer and set aside.

[0160] ② Sample addition: Set up standard sample wells and test sample wells separately. First add 40μl of serum sample diluent (5% BSA) to the test sample well, then add 10μl of the test sample. The sample should be added to the bottom of the ELISA plate well without touching the well wall. After adding, gently shake to mix; add BTD standard samples of different gradient concentrations to the standard sample wells, and the rest of the operation is the same as described above.

[0161] ③ Incubation: Seal the sample wells with sealing film and incubate at 37℃ for 30 minutes.

[0162] ④ Washing: Carefully peel off the sealing film, discard the liquid, and spin dry. Fill each well with washing solution (KH2PO4-Na2HPO4 buffer, pH=7.4), let it stand for 30 seconds and then discard it. Repeat this 5 times and pat dry.

[0163] ⑤ Add enzyme: add 50 μl of 1:500 horseradish peroxidase (HRP)-5% BSA enzyme labeling reagent to each well, incubate for 30 min, and wash with KH2PO4-Na2HPO4 buffer.

[0164] ⑥ Color development: first add 100 μl of 1 mg / ml TMB citric acid-Na2HPO4 buffer solution (pH=5.0) to each well, gently shake to mix, and incubate at 37°C in the dark for 15 minutes.

[0165] ⑦Termination: Add 50μl 2mmol / L H2SO4 to each well to terminate the reaction (the blue color immediately turns to yellow).

[0166] ⑧ Determination: Measure the absorbance (OD value) of each well at 450nm. The determination should be carried out within 15 minutes after adding the stop solution.

[0167] ⑨ Obtain standard curve: Establish a "concentration-absorbance" standard curve based on different concentration gradient standards and absorbance.

[0168] ⑩ Obtaining the serum BTD concentration of the sample to be tested: Detect the absorbance of BTD in the serum sample to be tested, and calculate the concentration of BTD based on the standard curve.

[0169] (4) Prediction of HFpEF

[0170] ① According to the screening criteria of (1), serum samples from 132 DM patients were obtained, including 72 patients with simple DM and 60 patients with DM and HFpEF.

[0171] ② According to the serum BTD concentration data of the DM group and the DM combined with HFpEF group obtained in steps (1) to (3), the ROC curve was prepared with "Sensitivity % (Sensitivity %)" as the ordinate and "100%-Specificity % (100%-Specificity %)" as the abscissa, and the AUC area of ​​BTD protein was calculated. The BTD concentration data of the DM group and the DM combined with HFpEF group were entered through the "Column" function of Graphpad Prism 8.0 (version 8.0.1) software, and the ROC curve option was clicked to obtain an AUC value of 0.8053 [0.7316, 0.8790], p < 0.001. The results were statistically significant, so serum BTD can be used as a biomarker to predict whether diabetic subjects develop HFpEF, with certain specificity, sensitivity and accuracy.

[0172] ③ According to the ROC data obtained by the software, click "Sensitivity & Specificity" to obtain the BTD concentration corresponding to a specific sensitivity and specificity. The cutoff value of BTD can be determined by the Youden index calculation formula to obtain the best screening effect. The calculation formula is:

[0173] Youden index = sensitivity + specificity - 1.

[0174] Through comparison, it was found that when the Youden index reached its maximum, the BTD concentration was 2.204nmol / L, so 2.204nM was used as the judgment boundary.

[0175] ④ Determination of prediction criteria: when the concentration range is 0-2.204nM, HFpEF is predicted to occur; when the concentration is greater than 2.204nM, HFpEF is predicted not to occur.

[0176] (5) Comparison of efficacy in predicting HFpEF in diabetic patients

[0177] Collect patient baseline data including BMI, gender, age, smoking and drinking habits, and medical history (hypertension, hyperlipidemia).

[0178] The key information of the H2FPEF scoring system was collected: BMI greater than 30 (+2 points), use of ≥2 antihypertensive drugs (+1 point), atrial fibrillation (+3 points), pulmonary artery pressure PASP>35 mmHg (+1 point), age>60 years (+1 point), E / e'>9 (+1 point), and the total score was calculated. The total score ≥6 was used as the basis for grouping (DM vs DM+HFpEF).

[0179] Key information of the HFA-PEFF scoring system was collected: a. septal e' < 7 cm / s or lateral e' < 10 cm / s or mean E / e' ≥ 15 or TR velocity > 2.8 m / s (+2 points); mean E / e' 9-14 or GLS < 16% (+1 point); b. LAVI > 34 ml / m 2 or LVMI ≥ 149 (male) 122 (female) g / m 2 And RWT>0.42(+2 points);29ml / m 2 <LAVI≤34ml / m 2 Or LVMI>115(male)95(female)g / m 2 or RWT>0.42 or left ventricular wall thickness ≥12mm (+1 point); c. In the case of sinus rhythm, NT-proBNP>220pg / mL or BNP>80pg / mL (+2 points); NT-proBNP is 125-220pg / mL or BNP is 35-80pg / mL (+1 point); c. In the case of atrial fibrillation, NT-proBNP>660pg / mL or BNP>240pg / mL (+2 points); NT-proBNP is 365-660pg / mL or BNP is 105-240pg / mL (+1 point). The total score was calculated, and the total score ≥5 was used as the basis for grouping (DM vs DM+HFpEF).

[0180] Comparison results: The results of the comparison of the effectiveness of detecting BTD levels, H2FPEF and HFA-PEFF in predicting the occurrence of HFpEF in diabetic patients are as follows Fig. 9 As shown in the figure. A is Elisa detection of plasma BTD content in patients with DM and DM+HFpEF; B is multivariate regression analysis to detect whether plasma BTD content, gender (female), age, BMI, hypertension, hyperlipidemia, smoking and drinking are independent factors affecting the occurrence of HFpEF in DM patients, with p<0.05 as the reference basis for independent factors; C is ROC curve detection of the sensitivity and specificity of three methods for predicting HFpEF: BTD, H2FPEF and HFA-PEFF.

[0181] Depend on Fig. 9 As shown in Figure A, compared with the DM group, the serum BTD content in patients with DM and HFpEF was significantly lower. The baseline data, disease history, and BTD concentration were entered into SPSS software (SPSS 25.0 version), and the multivariate logistic regression analysis module was used to analyze whether plasma BTD was an independent influencing factor for the occurrence of HFpEF in patients with DM. Fig. 9 As shown in B. Fig. 9 As shown in Figure B, after adjusting for gender, age, BMI, medical history (hypertension, hyperlipidemia) and lifestyle (smoking, drinking), BTD was still an independent factor protecting DM patients from HFpEF (OR = 0.656 [0.509, 0.753], p = 0.015). According to the serum BTD concentration data of the DM group and the DM combined with HFpEF group, the score data of the H2FPEF scoring system and the HFA-PEFF scoring system obtained from steps (1) to (3), the ROC curve was constructed with "Sensitivity % (Sensitivity %)" as the ordinate and "100%-Specificity % (100%-Specificity %)" as the abscissa, and the AUC area of ​​BTD protein was calculated. The BTD concentration data of the DM group and the DM combined with HFpEF group, the score data of the H2FPEF scoring system and the HFA-PEFF scoring system were entered through the "Column" function of GraphpadPrism 8.0 (version 8.0.1) software. Click the ROC curve option, and the AUC value of the H2FPEF scoring system for predicting HFpEF is 0.8303, with a 95% confidence interval of [0.7590, 0.9016], p<0.001; the AUC value of the HFA-PEFF scoring system for predicting HFpEF is 0.8201, with a 95% confidence interval of [0.7474, 0.8929], p<0.001; the AUC value of BTD is 0.8053, with a 95% confidence interval of [0.7316, 0.8790], p<0.001; as shown in the figure below: Fig. 9 As shown in C. Fig. 9As shown in Figure C, serum BTD can be used as a biomarker to predict whether diabetic subjects develop HFpEF, and its predictive effect is similar to that of the H2FPEF scoring system and the HFA-PEFF scoring system. Therefore, this scheme has certain specificity, sensitivity and accuracy.

[0182] Example 4

[0183] This embodiment provides an Elisa kit for predicting HFpEF in diabetic patients, as follows:

[0184] Basic principle: Based on the specific immune reaction between antigen and antibody, the blood sample of diabetic patients is mixed with horseradish peroxidase (HRP), and then a color reaction is produced by HRP and TMB substrate, and the absorbance at a wavelength of 450nm is measured, which is then used for the quantitative determination of serum BTD. The reagents and consumables included in the kit are shown in Table 1.

[0185] Table 1: Composition of Elisa kit for HFpEF detection in diabetic patients

[0186]

[0187] The Elisa kit of this embodiment is used to predict HFpEF in diabetic patients. The specific operation can refer to Example 2, which mainly includes four parts: sample collection; sample pretreatment; sample detection; sample analysis. Among them, the whole process of sample collection from preparation to sampling takes about 5 minutes; the whole process of sample pretreatment takes about 20 minutes; the sample detection takes about 2 hours; the sample analysis is estimated to be 10 minutes. Combining the above process, it is estimated that a single test will take about 2-3 hours. And according to the specifications of the well plate, 96-384 samples can be measured at one time, so the average time to obtain the results of each patient is about 0.5-2 minutes, which is much shorter than the 3-4 hours required for the results of a single patient in the aforementioned H2FPEF scoring system or HFA-PEFF scoring system.

[0188] In summary, the present invention has found through research that the sensitivity and specificity of biotinidase for predicting the occurrence of HFpEF in DM patients are significantly higher than other HFpEF subtypes, and preliminary cell experiments show that biotinidase has a significant protective effect on DM combined with HFpEF. Based on this, the present invention applies biotinidase to the preparation of reagents for detecting heart failure with preserved ejection fraction in diabetic patients. The detection reagent for heart failure with preserved ejection fraction in diabetic patients has the following advantages when used: (1) Good prediction effect, similar to the H2FPEF scoring system and the HFA-PEFF scoring system; (2) Short average time, multiple patients can be predicted at one time; (3) The average prediction cost of patients can be reduced; (4) The detection technology is simple, which can reduce the workload of medical staff and improve the efficiency of diagnosis and screening. Therefore, the use of biotinidase provided by the present invention in the preparation of reagents for detecting heart failure with preserved ejection fraction in diabetic patients is suitable for large-scale screening of heart failure with preserved ejection fraction in diabetic patients.

[0189] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. Use of biotinidase in the preparation of a reagent for detecting heart failure with preserved ejection fraction in diabetic patients; the heart failure with preserved ejection fraction in diabetic patients is the HFpEF-5 subtype.

2. The use according to claim 1, characterized in that: The reagents include a biotinidase standard.

3. The use according to claim 1, characterized in that: The reagents include biotinidase antibody.

4. The use according to claim 3, characterized in that: The biotinidase antibody is tagged with a fluorescent label.

5. The use according to claim 3, characterized in that: The reagent also includes a horseradish peroxidase-labeled secondary antibody and a color developing agent.

6. The use according to claim 5, characterized in that: The color developer is 3,3',5,5'-tetramethylbenzidine.

7. The use according to claim 1, characterized in that: The steps of using the reagent to detect heart failure with preserved ejection fraction in diabetic patients include: Collect whole blood from the subject to be tested, add anticoagulant, and obtain an initial sample; Pre-treating the initial sample to obtain a serum sample; detecting the concentration of biotinidase in the serum sample by using the reagent; The concentration of the biotinidase is used as a detection index to predict whether diabetic patients will develop heart failure with preserved ejection fraction.

8. The use according to claim 7, characterized in that: The anticoagulant is selected from one or more of ethylenediaminetetraacetic acid, sodium citrate, sodium citrate, and sodium heparin.

9. The use according to claim 7, characterized in that: The pretreatment is centrifugation or polymer material filtration.

10. The use according to claim 7, characterized in that: The prediction standard is: when the concentration of the biotinidase is between 0-2.204 nmol / L, it is predicted that heart failure with preserved ejection fraction will occur; when the concentration of the biotinidase is greater than 2.204 nmol / L, it is predicted that heart failure with preserved ejection fraction will not occur.

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