Application of biomarker combination in biological sample in preparation of vascular dementia detection product

Abstract

The invention discloses application of a biomarker combination in a biological sample to preparation of a vascular dementia detection product. The biomarker combination is prepared from phenylacetylurea, 3-methyl-2 (1H)-quinoxaline, pyruvic acid, L-cystine and 4-methylene glutamic acid. The applicant carries out regression analysis on the five metabolites and detects the expression level of the five metabolites between a healthy group and a disease group, the result shows that except the area of 4-methyleneglutamic acid under an AUC curve is 0.917, the areas of the other four metabolites under the AUC curve are all 1, extremely high specificity and sensitivity are shown, and the application prospect is wide. The method can be used for preparing related detection products for diagnosis or auxiliary diagnosis of vascular dementia.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to the application of a biomarker combination in a biological sample in the preparation of a vascular dementia detection product. Background Art

[0002] Vascular Dementia (VaD), as the second most common type of dementia, is usually a cognitive impairment syndrome caused by various cerebrovascular diseases (such as ischemic stroke, hemorrhagic stroke, and cerebrovascular diseases causing hypoperfusion in brain regions such as memory, cognition, and behavior). Its characteristic is the gradual decline of cognitive function, affecting memory, thinking, language, and behavioral abilities. The incidence of vascular dementia varies in different regions, and the incidence is relatively high in Asian countries, especially in China, which may be related to the relatively high incidence of stroke.

[0003] The treatment of vascular dementia mainly includes drug treatment and non-drug treatment. Drug treatment commonly uses cholinesterase inhibitors (such as donepezil) and NMDA receptor antagonists (such as memantine) to improve cognitive function, and at the same time combines antiplatelet drugs (such as aspirin) and antihypertensive drugs to prevent the recurrence of cerebrovascular events. Non-drug treatment focuses on cognitive training, lifestyle intervention (such as a healthy diet, exercise, and smoking cessation), and psychological support to delay cognitive decline and improve the quality of life. However, the existing treatment methods have limited effects and cannot completely prevent the progression of the disease. Therefore, the use of biomarkers for early diagnosis and monitoring is of great significance. Biomarkers (such as specific proteins in neuroimaging, blood, and cerebrospinal fluid) can help identify VaD at an early stage of the disease and achieve early intervention; at the same time, by dynamically monitoring biomarkers, the disease progression and treatment effect can be evaluated, providing a basis for individualized treatment and an important tool for new drug research and development and clinical trials, thereby improving the management and prognosis of VaD.

[0004] There are many serum markers currently under research, including homocysteine, C-reactive protein, apolipoprotein E4 (ApoE4), Tau protein, glial fibrillary acidic protein (GFAP), etc., and their specificity and sensitivity are not particularly ideal. The invention patent with publication number CN117741154A discloses the use of a biomarker composed of amyloid-β 1-42 / amyloid-β 1-40, amyloid-β 1-42, t-Tau protein, and phosphorylated tau181 protein in the preparation of a detection kit and / or reagent for mixed cortical and subcortical vascular dementia; the test results show that the sensitivity of this biomarker combination can reach 85.58%, the specificity can reach 91.11%, and the area under the ROC curve (AUC) can reach 0.913. At present, there is no relevant report on the use of phenacemide, 3-methyl-2(1H)-quinoxaline, pyruvate, L-cystine, and 4-methyleneglutamic acid as a biomarker combination for the diagnosis or monitoring of vascular dementia. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an application of a biomarker combination in a biological sample in the preparation of a detection product for vascular dementia, aiming at the insufficient specificity and sensitivity of biomarkers in the detection of vascular dementia in the prior art.

[0006] To solve the above technical problems, the present invention adopts the following technical solutions:

[0007] An application of a biomarker combination in a biological sample in the preparation of a detection product for vascular dementia, wherein the biomarker combination is composed of phenacemide (CAS No.: 63-98-9), 3-methyl-2(1H)-quinoxaline (CAS No.: 14003-34-0), pyruvate (CAS No.: 127-17-3), L-cystine (CAS No.: 56-89-3), and L-2-amino-4-methylenepentanedioic acid (CAS No.: 7150-74-5).

[0008] The detection product can be a kit and / or a reagent.

[0009] The vascular dementia includes early vascular cognitive impairment, mid-late and severe vascular dementia.

[0010] The test results of the applicant show that when the above biomarker combination is applied to the detection of patients with vascular dementia, compared with the control group (normal healthy population), the concentration of the biomarker L-cysteine in the biological samples of patients with vascular dementia increases, while the concentrations of the remaining biomarkers (phenylacetylurea, 3-methyl-2(1H)-quinoxaline, pyruvic acid, and 4-methyleneglutamic acid) decrease. Therefore, when the concentration of L-cysteine among the aforementioned biomarkers in the biological sample of a subject is up-regulated compared with the control group, and the concentration of one or more of the remaining biomarkers is down-regulated compared with the control group, it can be used to diagnose or assist in diagnosing that the subject providing the biological sample has vascular dementia or is at risk of developing vascular dementia.

[0011] In some embodiments, the level of the biomarker in the biological sample can be determined by the following methods: chromatography and / or mass spectrometry, fluorometry, electrophoresis, immunoaffinity, hybridization, immunochemistry, ultraviolet spectroscopy, fluorescence analysis, radiochemical analysis, near-infrared spectroscopy, nuclear magnetic resonance spectroscopy, light scattering analysis.

[0012] In some preferred embodiments, the level of the biomarker in the biological sample is determined by spectroscopy, liquid or gas chromatography, mass spectrometry, liquid or gas chromatography coupled with mass spectrometry.

[0013] When mass spectrometry is used for detection, a full scan mode is employed for screening combined with second-level targeted analysis. The full scan mode collects all primary information of small molecules within the mass range of 50 m / z to 1200 m / z in the biological sample of the subject. Potential biomarkers are screened as differential metabolites through multivariate statistical analysis, and the potential biomarkers are further subjected to targeted second-level fragmentation. Combining with the second-level spectra in the database, the differential molecules are finally determined as the biomarkers described in the present invention.

[0014] The biological sample can be whole blood, serum, or plasma from the subject. In some preferred embodiments, the biological sample is serum. The subject is a mammal, such as a human.

[0015] The present invention also includes a detection product for diagnosing or assisting in diagnosing vascular dementia by a biomarker combination in a biological sample, wherein the biomarker combination consists of phenylacetylurea, 3-methyl-2(1H)-quinoxaline, pyruvic acid, L-cysteine, and 4-methyleneglutamic acid. The detection product is a kit and / or reagent. In a preferred embodiment, the detection product, in addition to containing the above biomarker combination, also contains optional internal standards and / or metabolite extraction reagents.

[0016] Compared with the prior art, the present invention provides an application of a biomarker combination in a biological sample in the preparation of a detection product for diagnosing or assisting in the diagnosis of vascular dementia. The inventor team of the present application has creatively discovered that 5 metabolites are obtained from the blood of vascular dementia patients by liquid chromatography-high resolution mass spectrometry as biomarkers for preparing relevant detection products for diagnosing or assisting in the diagnosis of vascular dementia. Regression analysis was performed on these 5 metabolites and their expression levels between the healthy group and the disease group were detected. The results showed that the area under the AUC curve of 4 metabolites was 1, and the area under the AUC curve of 4-methyleneglutamic acid was 0.917, showing extremely high specificity and sensitivity. Description of the Drawings

[0017] Figure 1 It is the PLS-DA score plot of the principal component analysis of the serum metabolic profiles of the experimental group and the control group in Example 1, where Patients represents the disease group and Healthy represents the healthy group.

[0018] Figure 2 It is the result of the cluster heat map analysis of the serum differential metabolites of the experimental group and the control group in Example 1.

[0019] Figure 3 It is the result of the exploratory analysis of the ROC curve of the multivariate serum differential metabolites of the experimental group and the control group in Example 1.

[0020] Figure 4 It is the top 10 metabolite plot of the exploratory analysis of the ROC curve of the multivariate serum differential metabolites of the experimental group and the control group in Example 1 under the best mode.

[0021] Figures 5A to 5E They are respectively the ROC curves of the serum differential metabolites phenylacetylurea, 3-methyl-2(1H)-quinoxaline, pyruvic acid, L-cysteine, 4-methyleneglutamic acid of the experimental group and the control group in Example 1 and their expression levels in each group. Detailed Embodiments

[0022] The present invention will be further described below with reference to the drawings through examples, but it is not intended to limit the present invention. The specific materials used in the embodiments of the present invention and their sources are provided below. However, it should be understood that these are merely exemplary and are not intended to limit the present invention. Materials with the same or similar types, models, qualities, properties or functions as the following reagents and instruments can be used to implement the present invention. The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0023] Screening of differential serum metabolic markers for vascular dementia and evaluation of diagnostic efficacy

[0024] 1. Preparation of metabolite samples:

[0025] 1.1 Source of clinical samples: Six patients with vascular dementia who were hospitalized in the Department of Neurology of the Affiliated Hospital of Guilin Medical University from December 2019 to June 2021 were selected as the experimental group (disease group). Experienced neurosurgeons collected medical histories and performed physical examinations and finally made a definite diagnosis. Six healthy subjects undergoing physical examinations in our hospital during the same period were selected as the control group (healthy group). There were 3 males and 3 females in the experimental group, aged between 48 and 78 years old. There were 3 males and 3 females in the control group, aged between 31 and 56 years old.

[0026] 1.2 Collection and processing of samples: Fasting venous blood was drawn and centrifuged at 3000g for 5 minutes, and the supernatant was taken as the serum sample. 200 μL of acetonitrile was added to 50 μL of serum and vortexed, and then centrifuged at 4°C and 12000 rpm for 10 minutes. The supernatant was transferred to a 1.5 ml centrifuge tube. The supernatant was dried by a centrifugal concentrator. It was reconstituted with 100 μL of 1% acetonitrile, and the supernatant was taken for testing.

[0027] 2. Mass spectrometry detection of samples

[0028] 2.1 Instrument conditions: One in one hundred thousand electronic analytical balance (Sartorius Practum, SQP type, Sartorius Scientific Instruments (Beijing) Co., Ltd.); Vortex mixer (Vortex-Genie2, Scientific Industries, USA); Centrifugal concentrator (Genevac miVac, Tegent Scientific Ltd., UK); The instrument used an ultra-high performance liquid chromatograph (Agilent 1290II, Agilent Technologies, Germany) in series with a high-resolution mass spectrometer (5600 Triple TOFPlus, AB Sciex, Singapore); All analyses were carried out in the electrospray ionization (ESI±) mode, and the conditions were as follows: curtaingas = 35, ion spray voltage = 5500V in positive ion mode, ion spray voltage = -4500V in negative ion mode, temperature = 450°C, ion source gas 1 = 50, ion source gas 2 = 50.

[0029] 2.2 Data acquisition mode: TOF full scan for the first-level mass spectrometry; information-dependent acquisition (IDA) for the second-level mass spectrometry; collision energy (±) 30 ± 15 eV; internal standards include: Phenylalanine-D8, Tryptophan-D8, Isoleucine-D10, Asparagine-13C4, Methionine-D3, Valine-D8, Proline-D7, Alanine-D4, Glycine-D2, Serine-D3, Glutamate-D5, Aspartate-D3, Arginine-D7, Glutamine-D5, Lysine-D9, Histidine-D5, D13-choline, (13C,15N3)-Uric acid, (15N)4-Inosine, D5-Benzoic acid, D11-Betaine, P-cresol sulfate-D7, etc.

[0030] 2.3 Data processing: The original data peak area, mass-to-charge ratio, and retention time of the first-level mass spectrometry were extracted by MarkerView 1.3 (AB Sciex, Concord, ON, Canada) software to generate a two-dimensional data matrix (filtering out isotope peaks); PeakView 2.2 (AB Sciex, Concord, ON, Canada) was used to extract the second-level mass spectrometry data and compare it with the Metabolites database, HMDB, METLIN, and reference standards to identify metabolite IDs, and the identified IDs were assigned to the corresponding ions in the two-dimensional data matrix of the first-level mass spectrometry. A self-written program based on the R language was used to statistically analyze and perform pathway analysis on the identified metabolomics data, and finally, the serum metabolites of the control group and the experimental group and the levels of metabolites in each sample were obtained.

[0031] 3. Analyze and screen out blood metabolomics markers for vascular dementia

[0032] The obtained serum metabolites were analyzed by principal component analysis (PCA) and combined with orthogonal partial least squares discriminant analysis (PLS-DA) to construct a model for analysis. The results showed that there was a significant difference between the control group and the experimental group, and the results were as Figure 1 shown.

[0033] In addition to principal component analysis, heatmaps are also often used in omics research to observe the overall differences in samples, which can facilitate the discovery of outliers. Therefore, we analyzed the obtained serum metabolites by correlation heatmap, and found that the differential changes of multiple metabolites were positively or negatively correlated with vascular dementia. The results are shown in Figure 2 as follows.

[0034] Therefore, we further explored which serum metabolites might be potential biomarkers for vascular dementia through ROC curve exploratory analysis. Using the Biomarker Analysis tool in the MetaboAnalyst (https: / / www.metaboanalyst.ca / ) metabolite database, we could see that the best prediction results could be obtained with a combination of 5 metabolites in the optimal mode. The area under the AUC curve of the 5 differential metabolite combinations was 0.81, and the confidence interval CI was 0.056 - 1. The results are shown in Figure 3 as follows.

[0035] We further analyzed which serum metabolites were key under this optimal model. The analysis results showed that the top 10 key metabolites are shown in Figure 4 as follows. We selected a combination of 5 metabolites, namely Phenacemide, 3-methyl-2(1H)-Quinoxali, Pyruvate, L-Cystine, and L-2-Amino-4-methylenepentanedioic acid, as potential biomarkers for vascular dementia for further analysis. The results are shown in Figure 4 as follows.

[0036] Finally, we performed a regression analysis on these 5 selected serum metabolites and detected their expression levels between the control group and the experimental group. The results showed that the area under the AUC curve of all 4 metabolites except L-2-Amino-4-methylenepentanedioic acid was 1, showing extremely high specificity and sensitivity. In addition, the levels of the 4 differential metabolites, namely Phenacemide, 3-methyl-2(1H)-Quinoxali, Pyruvate, and L-2-Amino-4-methylenepentanedioic acid, were downregulated after vascular dementia, while the level of L-Cystine was upregulated. The results are shown in Figures 5A to 5E as follows.

[0037] According to the above experimental results, the combination of 5 serum metabolites, namely phenacetylurea, 3-methyl-2(1H)-quinoxaline, pyruvic acid, 4-methyleneglutamic acid and L-cysteine, has strong sensitivity and specificity, is suitable as a blood metabolite marker for the diagnosis or monitoring of vascular dementia, and can be used to prepare related detection products for the diagnosis or auxiliary diagnosis of vascular dementia.

[0038] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of a biomarker combination in a biological sample in the preparation of a vascular dementia detection product, wherein the biomarker combination consists of phenylacetyl urea, 3-methyl-2(1H)-quinoxaline, pyruvic acid, L-cystine and 4-methyleneglutamate.

2. The use according to claim 1, characterized in that: The detection product is a kit and / or a reagent.

3. The use according to claim 1, characterized in that: The level of the biomarker in the biological sample is measured by the following methods: chromatography and / or mass spectrometry, fluorescence measurement, electrophoresis, immunoaffinity, hybridization, immunochemistry, ultraviolet spectroscopy, fluorescence analysis, radiochemical analysis, near infrared spectroscopy, nuclear magnetic resonance spectroscopy, light scattering analysis.

4. The use according to claim 1, characterized in that: The level of the biomarker in the biological sample is measured by spectroscopy, liquid or gas chromatography, mass spectrometry, or liquid or gas chromatography coupled to mass spectrometry.

5. The use according to any one of claims 1 to 4, characterized in that: The biological sample is selected from whole blood, serum or plasma.

6. A detection product for diagnosing or assisting in the diagnosis of vascular dementia by combining biomarkers in a biological sample, characterized in that: The biomarker combination consists of phenylacetyl urea, 3-methyl-2(1H)-quinoxaline, pyruvate, L-cystine and 4-methyleneglutamate.

7. The detection product according to claim 6, characterized in that: The detection product is a kit and / or a reagent.

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