CN115808492A - Alzheimer's disease biomarker asparagine and application thereof - Google Patents
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Abstract
The invention relates to an Alzheimer's disease biomarker asparagine and application thereof. The biomarker for alzheimer's disease is asparagine. According to the invention, the level of asparagine in the blood sample of Alzheimer's disease is obviously lower than that of a normal blood sample for the first time, asparagine in blood is taken as a biomarker of Alzheimer's disease, early diagnosis of Alzheimer's disease can be assisted by detecting the level of asparagine in blood, noninvasive and rapid detection is facilitated, and the method has the characteristics of timeliness, convenience, high specificity and high sensitivity.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to an asparagine as a biomarker of Alzheimer's disease and application thereof.
Background
Alzheimer's Disease (AD), also known as senile dementia, is a progressive degenerative disease of the central nervous system occurring in the elderly, characterized by progressive memory impairment, cognitive decline and loss of daily life, accompanied by psychogenic symptoms such as personality changes, which seriously affect social and life functions, and because the pathogenesis of Alzheimer's disease is not completely clear, and its early symptoms are more secret, patients with Alzheimer's disease are easily missed or misdiagnosed.
The current early screening technologies for AD comprise Positron Emission Tomography (PET), cerebrospinal fluid Abeta molecular level detection and the like, wherein the former needs to inject a certain dose of radioactive substances into a detected person, the latter has large operation damage and is easy to cause surgical infection, and the reliability of the diagnosis technology for AD early diagnosis is unstable, so that the diagnosis technology is difficult to be used for AD early screening.
Therefore, the development of new markers for early diagnosis of AD is one of the important directions for AD diagnosis and treatment in the future.
CN112858684A discloses a neurodegenerative disease marker Chromogranin B and application thereof, wherein the marker comprises phosphorylation Chromogranin B protein, 9 peptide segments of the Chromogranin B protein are phosphorylated to serve as markers of neurodegenerative diseases, the markers are used for judging and evaluating symptoms of the neurodegenerative diseases such as AD and PD, and the constructed kit sample is simple in early-stage treatment process, small in sample consumption and high in accuracy, and has important clinical guiding significance for auxiliary diagnosis of related indexes of AD and PD.
With the development of high-throughput omics technologies such as genomics, transcriptomics, proteomics and metabonomics, the research and development of novel biomarkers, particularly metabonomics, are accelerated, the dynamic change of metabolite profiles is monitored by the technologies such as magnetic resonance spectrum and mass spectrum, the dynamic change-monitoring biomarker has great advantages in screening disease-related biomarkers, blood contains proteins, polypeptides, nucleic acids, lipids and other metabolites, blood samples are convenient to obtain and have small invasiveness, and the dynamic change-monitoring biomarker has great application potential in screening biomarkers.
In conclusion, the new blood metabolite-based AD biomarker is screened, so that the judgment basis of AD early diagnosis can be expanded, the AD biomarker can be combined with other marker detection, the AD diagnosis accuracy is improved, and early warning, pathological typing, prediction and evaluation of development stages and the like of diseases are facilitated.
Disclosure of Invention
Aiming at the defects and actual requirements of the prior art, the invention provides an Alzheimer's disease biomarker asparagine and application thereof, the invention carries out qualitative and quantitative analysis on metabolites in human blood based on a high-resolution non-targeted metabonomics analysis technology, firstly takes asparagine in the blood as an Alzheimer's disease marker, can assist early diagnosis of Alzheimer's disease by detecting the level of asparagine in the blood, and has the characteristics of timeliness, convenience, high specificity and high sensitivity.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, a biomarker of alzheimer's disease, said biomarker of alzheimer's disease is Asparagine (L-Asparaginine).
Asparagine is an amino acid with the molecular formula C 27 H 18 C l3 N 3 O, molecular weight 506.8103, can be used as medicine for lowering blood pressure, dilating bronchus (relieving asthma), resisting peptic ulcer and gastric dysfunction, and can also be used for microorganism culture, acrylonitrile sewage treatment, etc.
According to the invention, qualitative and quantitative analysis is carried out on blood metabolites based on a high-resolution non-targeted metabonomics analysis technology, the asparagine level in the blood sample of the Alzheimer disease is obviously lower than that of a normal blood sample, the asparagine in the blood is used as a biomarker of the Alzheimer disease, and the early diagnosis of the Alzheimer disease can be assisted by detecting the asparagine level in the blood.
The invention provides application of asparagine as a biomarker in assisting early diagnosis of Alzheimer's disease.
In a second aspect, the present invention provides the use of the biomarker for alzheimer's disease as described in the first aspect in constructing an early diagnosis model for alzheimer's disease and/or in preparing an early diagnosis device for alzheimer's disease.
In a third aspect, the present invention provides an early diagnosis model of alzheimer's disease, wherein the input variables of the early diagnosis model of alzheimer's disease comprise the peak intensity values of the asparagine mass spectrum of the first aspect.
Preferably, the output variables of the model for early diagnosis of alzheimer's disease comprise fold differential expression, and the calculation formula of the fold differential expression is shown in equation (1):
preferably, the positive judgment standard of the Alzheimer's disease is that the differential expression multiple is less than or equal to 0.64.
According to the invention, the peak intensity values of the asparagine mass spectrum in the normal blood sample and the AD blood sample are fully compared and analyzed, and rational design is carried out, so that the model for early diagnosis of the Alzheimer's disease is constructed, the model takes the peak intensity value of the asparagine mass spectrum as an input variable, takes the differential expression multiple as an output variable, can rapidly output a result, and fully represents the samples with abnormal asparagine level, thereby assisting the early diagnosis of the Alzheimer's disease.
In a fourth aspect, the present invention provides an apparatus for early diagnosis of alzheimer's disease, comprising:
a sample preparation unit: preparing a sample to be detected into a sample solution to be detected which can be used for the separation of a liquid chromatograph;
a detection unit: separating the sample solution to be detected by using the liquid chromatograph, performing data processing on the separated sample by using a mass spectrometer, and determining the peak intensity value of the asparagine mass spectrum in the first aspect in the sample;
an analysis unit: inputting the peak intensity value of the detected asparagine mass spectrum into the early diagnosis model for Alzheimer disease described in the third aspect for analysis;
an evaluation unit: and outputting the differential expression multiple corresponding to the sample, and judging whether the sample is positive for the Alzheimer's disease.
In the device for early diagnosis of Alzheimer's disease, all units are effectively matched, the device is simple and efficient, sample processing, detection and differential expression multiple obtaining can be rapidly completed, positive assessment of Alzheimer's disease is carried out according to a reasonably designed judgment standard, and the device has important significance for early diagnosis of Alzheimer's disease.
Preferably, the sample to be tested comprises a blood sample.
Preferably, the preparation method of the sample solution to be detected comprises the steps of adding a sample to be detected into an acetonitrile aqueous solution, centrifuging and collecting supernatant, so as to obtain the sample solution to be detected.
Preferably, the preparation method of the sample solution to be tested comprises the following steps:
(1) Adding a sample to be detected into a precooled methanol/acetonitrile/water solution, mixing and ultrasonically standing for 5-15 min (such as 26min, 27min, 28min, 29min or 32 min) at-20 to-15 ℃ (such as-19 ℃, -18 ℃, -16 ℃ or-17 ℃), such as 6min, 7min, 8min, 9min, 10min, 12min or 14 min), centrifuging for 15-25 min (such as 16min, 17min, 18min, 19min, 20min, 21min, 22min, 23min or 24 min) at 0-4 ℃ (such as1 ℃, 2 ℃ or 3 ℃), such as 12200 × g, 00 × g, 12600 × g, 12800 × g, 13200 × g, 12600 × g, 15000 × g or 15800 × g), and taking the supernatant to vacuum dry to obtain a pretreated sample;
(2) Adding the pretreated sample into 80-120 mu L acetonitrile aqueous solution for redissolving, vortexing, centrifuging at 0-4 ℃ and 12000-16000 Xg (for example, 12200 Xg, 12400 Xg, 12600 Xg, 12800 Xg, 13200 Xg, 12600 Xg, 15000 Xg or 15800 Xg) for 10-20 min (for example, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min or 19 min), and taking supernatant to obtain the sample solution to be tested.
Preferably, the volume ratio of methanol, acetonitrile and water in the methanol/acetonitrile/water solution is (1-2): 1 includes but is not limited to 1.2.
Preferably, the volume ratio of acetonitrile to water in the aqueous acetonitrile solution is (1-2) 1, including but not limited to 1.1.
Preferably, the liquid chromatograph comprises an ultra high performance liquid chromatograph.
Preferably, the ultra high performance liquid chromatograph comprises an Agilent1290infinityl lc ultra high performance liquid chromatograph.
Preferably, the mass spectrometer comprises a tandem time-of-flight mass spectrometer.
Preferably, the tandem time-of-flight mass spectrometer comprises an ABTripleTOF6600 mass spectrometer.
Preferably, the data processing comprises:
collecting a primary spectrogram and a secondary spectrogram of the separated sample by using a series time-of-flight mass spectrometer, converting the primary spectrogram and the secondary spectrogram into an mzXML format, then performing peak alignment, retention time correction, peak area extraction and structure identification, and determining the peak intensity value of the asparagine mass spectrum of the first aspect in the sample.
Preferably, the alzheimer's disease early diagnosis device includes:
a sample preparation unit: preparing a sample to be detected into a sample solution to be detected which can be used for the separation of a liquid chromatograph;
a detection unit: separating the sample solution to be detected by using the liquid chromatograph, collecting a primary spectrogram and a secondary spectrogram of the separated sample by using a tandem time-of-flight mass spectrometer, converting the primary spectrogram and the secondary spectrogram into an mzXML format, performing peak alignment, retention time correction, peak area extraction and structure identification, and determining the peak intensity value of the asparagine mass spectrum of the first aspect in the sample;
an analysis unit: inputting the peak intensity value of the detected asparagine mass spectrum into the early diagnosis model for Alzheimer disease described in the third aspect for analysis;
an evaluation unit: and outputting the differential expression multiple corresponding to the sample, and judging whether the sample is positive for the Alzheimer's disease.
In the invention, the detection of the asparagine level in the blood sample can be used as a diagnosis basis, and can be combined with other detection results to assist the early diagnosis of the Alzheimer's disease, so that the accuracy of the diagnosis of the Alzheimer's disease can be expected to be improved, but the detection can not be used alone as a diagnosis index capable of 100% diagnosis of the Alzheimer's disease.
In the present invention, L-Asparagine was found to belong to the biosynthetic pathway of amino acids (Biosynthesis of amino acids pathway) by KEGG pathway analysis.
In a fifth aspect, the present invention provides the use of the biomarker for alzheimer's disease described in the first aspect in screening a medicament for treating and/or preventing alzheimer's disease.
Namely, the Alzheimer's disease biomarker of the first aspect is used as a target for screening drugs for treating and/or preventing Alzheimer's disease.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, the level of asparagine in the blood sample of Alzheimer's disease is obviously lower than that of a normal blood sample for the first time, asparagine in blood is taken as a biomarker of Alzheimer's disease, early diagnosis of Alzheimer's disease can be assisted by detecting the level of asparagine in blood, noninvasive and rapid detection is facilitated, and the method has the characteristics of timeliness, convenience, high specificity and high sensitivity.
Drawings
FIG. 1 is a graph of asparagine levels in blood samples of AD model mice and wild type mice;
FIG. 2 is a graph of histidine levels in cerebral cortex samples from AD model mice and wild type mice;
FIG. 3 is a graph of glycine levels in cerebral cortex samples of AD model mice and wild type mice;
FIG. 4 is a graph of dihydroxyacetone phosphate levels in cerebral cortex samples from AD model mice and wild type mice;
FIG. 5 is a graph of D-erythrose-4-phosphate levels in cerebral cortex samples of AD model mice and wild type mice;
FIG. 6 is a graph of phosphoenolpyruvate levels in cerebral cortex samples from AD model mice and wild type mice;
FIG. 7 is a graph of the level of 3-phosphoglycerate in cerebral cortex samples from AD model mice and wild type mice.
Detailed Description
To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
Example 1
This example provides qualitative and quantitative metabolite analysis of blood samples from 9-month-old AD model mice (APP/PS 1 transgenic mice, provided by the university of Nanjing model animal research institute) and Wild Type (WT) mice.
Respectively collecting blood of 10 AD model mice and 10 wild type mice cultured under the same condition, sequentially numbering the wild type mouse samples as SWT-1-SWT-1-10, sequentially numbering the AD model mouse samples as STG-1-STG-1-10, and detecting the asparagine level in the samples by adopting ultra-performance liquid chromatography-tandem flight time mass spectrometry, wherein the specific method comprises the following steps:
(1) Adding a blood sample into a precooled methanol/acetonitrile/water solution (volume ratio is 2;
(2) Adding the pretreated sample into 100 mu L acetonitrile aqueous solution (volume ratio is acetonitrile: water = 1), redissolving, vortexing, centrifuging at 4 ℃ and 14000 Xg for 15min, taking supernatant, injecting and analyzing, and separating by using an Agilent1290Infinity LC ultra-high performance liquid chromatography system (UHPLC) HILIC chromatographic column with the column temperature of 25 ℃; the flow rate is 0.5mL/min; the sample volume is 2 mu L; the mobile phase composition comprises: phase A: mixed aqueous solution of ammonium acetate and aqueous ammonia (both ammonium acetate and aqueous ammonia at 25mM final concentration), phase B: acetonitrile; the gradient elution procedure was as follows: 0 to 0.5min,95% of phase B; 0.5-7min, phase B changed linearly from 95% to 65%; 7-8min, wherein the phase B is linearly changed from 65% to 40%; 8-9min, and maintaining the phase B at 40%; 9-9.1min, the phase B changes from 40% to 95% linearly; 9.1-12min, and maintaining the phase B at 95%; the sample is placed in an automatic sample injector at 4 ℃ in the whole analysis process;
(3) And (3) collecting primary and secondary spectrograms of the sample separated by the ultra-high performance liquid chromatography system in the step (2) by adopting an AB Triple TOF6600 mass spectrometer, wherein ESI source conditions are as follows: ion Source Gas1 (Gas 1): 60, ion Source Gas2 (Gas2): 60, curtain gas (CUR): 30, source temperature: ionSapary Voltage flowing (ISVF) + -5500V (positive and negative modes) at 600 ℃; TOF MS scan m/z range:60-1000Da, product ion scan m/z range:25-1000Da, TOF MS scan accumulation time 0.2 s/spectra, product ion scan accumulation time 0.05s/spectra; secondary mass spectra were acquired using Information Dependent Acquisition (IDA) and high sensitivity mode, statistical potential (DP): ± 60V (positive and negative modes), fusion Energy: 35. + -.15eV, IDA the following extract isotopes with 4Da, candidates ions to monitor per cycle:10, converting the collected raw data in the Wiff format into an mzXML format through a Proteo Wizard, performing peak alignment, retention time correction and peak area extraction by using an XCMS software, performing metabolite structure identification on the data extracted by the XCMS, analyzing the asparagine level in a sample, and performing Fold variation Analysis (FC Analysis), principal Component Analysis (PCA), orthogonal partial least squares discriminant Analysis (OPLS-DA) and T-test (Student's T-test) by using an R language tool (R packages), wherein the results are shown in fig. 1 and table 1, the asparagine level in the blood sample of the AD model mouse is significantly lower than that of a wild type mouse, which indicates that asparagine in the blood can be used as an alzheimer biomarker, and early diagnosis of alzheimer's disease can be assisted by detecting the asparagine level in the blood.
TABLE 1
| Metabolites | VIP | Fold differential expression | p-value | Significance of | |
| AD model mouse vs wild type mouse | Asparagine | 1.02 | 0.64 | 0.035 | * |
Note: * P <0.05.
In addition, through the annotation and analysis of KEGG pathway, asparagine is found to belong to the biosynthetic pathway of amino acids (Biosynthesis of amino acids pathway), and is one of the 20 most common amino acids, namely, an amino acid with amide group, which is generated by transamination of aspartic acid, can be converted into aspartic acid under the catalysis of asparaginase, is related to the development of brain function, plays an important role in the systemic ammonia cycle, and has the functions of regulating the body metabolism and improving the human immunity.
Example 2
This example provides qualitative and quantitative analysis of metabolites in cerebral cortex samples of 9-month-old AD model mice and Wild Type (WT) mice.
Respectively collecting cerebral cortex samples of 10 AD model mice and 10 wild type mice cultured under the same condition, sequentially numbering the wild type cerebral cortex samples as CWT-1-CWT-1-10, sequentially numbering the cerebral cortex samples of the AD model mice as CTG-1-CTG-1-10, and performing qualitative and quantitative analysis on metabolites by adopting an ultra-high performance liquid chromatography-tandem flight time mass spectrometer, wherein the specific method comprises the following steps:
(1) Cutting open the brain shell of a mouse by using a surgical scissors after the neck of the mouse is cut off, exposing the brain, cutting the brain into two halves along the middle brain seam, respectively removing the cerebellum, the brainstem, the thalamus, the hypocortex and the hippocampus, leaving the cerebral cortex, collecting the complete cerebral cortex on the left and right sides as a sample, adding precooled methanol/acetonitrile/water solution (volume ratio is 2;
(2) Adding the pretreated sample into 100 mu L of acetonitrile aqueous solution (volume ratio is acetonitrile: water = 1) for redissolution, vortexing, centrifuging at 4 ℃ and 14000 Xg for 15min, taking supernatant, injecting and analyzing, and separating by using an Agilent1290Infinity LC ultra-performance liquid chromatography system (UHPLC) HILIC chromatographic column under the same conditions as in example 1;
(3) An AB Triple TOF6600 mass spectrometer is adopted to collect primary and secondary spectrograms of the sample separated by the ultra performance liquid chromatography system in the step (2), the conditions are the same as those in the embodiment 1, the collected raw data in the Wiff format is converted into an mzXML format through Proteo Wizard, then XCMS software is adopted to carry out peak alignment, retention time correction and peak area extraction, the data extracted by XCMS is subjected to metabolite structure identification, and then univariate statistical analysis, multidimensional statistical analysis, differential metabolite screening, differential metabolite correlation analysis and KEGG channel analysis are carried out, wherein Variable weighting values (Variable impedance for the project, VIP) obtained by an OPLS-DA model can be used for measuring the influence strength and the interpretation capability of the expression mode of each metabolite on the classification and discrimination of each group of samples, the present example, which considers VIP value and p-value together to screen significant differential metabolites, revealed that as shown in FIG. 2-FIG. 7 and Table 2, in AD model mice cerebral cortex tissues, levels of histidine (L-histidol), glycine (Glycine), dihydroxyacetone phosphate (Dihydroxyacetone phosphate) and D-Erythrose 4-phosphate (D-erythose 4-phosphate) were significantly higher than those of wild type mice, levels of Phosphoenolpyruvate (Phosphoenolpyruvate) and 3-phosphoglycerate (3-Phospho-D-glycerate) were significantly lower than those of wild type mice, and the above metabolites all belong to the biosynthetic pathway of amino acids (biosynthetic of amino acids pathway), indicating that the biosynthetic pathway of amino acids (biosynthetic of amino acids pathway) is associated with Alzheimer's disease, asparagine also belongs to the biosynthetic pathway of amino acids, and the change of asparagine level in blood metabolites is shown from another aspect to possibly reflect the abnormality of related metabolic pathways in AD brain, thereby having important significance for clinical early diagnosis.
TABLE 2
Note: * P <0.05, p <0.01.
In conclusion, according to the invention, the asparagine level in the blood sample of the Alzheimer's disease is detected to be significantly lower than that of the normal blood sample for the first time, the asparagine level in the blood is used as the biomarker of the Alzheimer's disease, the early diagnosis of the Alzheimer's disease can be assisted by detecting the asparagine level in the blood, the noninvasive rapid detection is facilitated, and the characteristics of timeliness, convenience, high specificity and high sensitivity are realized.
The applicant states that the present invention is illustrated by the above examples to show the detailed method of the present invention, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be carried out. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (10)
1. An alzheimer's disease biomarker, wherein the alzheimer's disease biomarker is asparagine.
2. The use of the biomarker of alzheimer's disease according to claim 1 for constructing an early diagnosis model of alzheimer's disease and/or for preparing an early diagnosis device of alzheimer's disease.
3. An early diagnosis model of Alzheimer's disease, wherein the input variables of the early diagnosis model of Alzheimer's disease comprise the peak intensity values of the asparagine mass spectrum of claim 1.
4. The model for early diagnosis of Alzheimer's disease according to claim 3, wherein the output variables of the model for early diagnosis of Alzheimer's disease comprise fold differential expression, which is calculated according to the formula shown in equation (1):
5. the model of claim 4, wherein the positive assessment criteria for Alzheimer's disease is that the fold difference between the expression levels is 0.64 or less.
6. An early diagnosis device for Alzheimer's disease, comprising:
a sample preparation unit: preparing a sample to be detected into a sample solution to be detected which can be used for the separation of a liquid chromatograph;
a detection unit: separating the sample solution to be detected by using the liquid chromatograph, processing data of the separated sample by using a mass spectrometer, and determining the peak intensity value of the asparagine mass spectrum of claim 1 in the sample;
an analysis unit: inputting the peak intensity values of the detected asparagine mass spectrum into the alzheimer's disease early diagnosis model according to any one of claims 3-5 for analysis;
an evaluation unit: and outputting the differential expression multiple corresponding to the sample, and judging whether the sample is positive for the Alzheimer's disease.
7. The device of claim 6, wherein the test sample comprises a blood sample.
8. The apparatus of claim 6 or 7, wherein the data processing comprises:
collecting a primary spectrogram and a secondary spectrogram of the separated sample by using a tandem time-of-flight mass spectrometer, converting the primary spectrogram and the secondary spectrogram into an mzXML format, performing peak alignment, correction of retention time, extraction of peak area and structure identification, and determining the peak intensity value of the asparagine mass spectrum of claim 1 in the sample.
9. The device according to any one of claims 6-8, characterized in that it comprises the following units:
a sample preparation unit: preparing a sample to be detected into a sample solution to be detected which can be used for the separation of a liquid chromatograph;
a detection unit: separating the sample solution to be detected by using the liquid chromatograph, collecting a primary spectrogram and a secondary spectrogram of the separated sample by using a tandem time-of-flight mass spectrometer, converting the primary spectrogram and the secondary spectrogram into an mzXML format, performing peak alignment, retention time correction, peak area extraction and structure identification, and determining the peak intensity value of the asparagine mass spectrum of claim 1 in the sample;
an analysis unit: inputting the peak intensity values of the detected asparagine mass spectrum into the Alzheimer's disease early diagnosis model of any one of claims 3-5 for analysis;
an evaluation unit: and outputting the differential expression multiple corresponding to the sample, and judging whether the sample is positive for the Alzheimer's disease.
10. Use of the biomarker for alzheimer's disease according to claim 1 for screening of a medicament for the treatment and/or prevention of alzheimer's disease.
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| WO2008021515A2 (en) * | 2006-08-18 | 2008-02-21 | Huntington Medical Research Institutes | Methods of determining levels of free amino acids and dipeptides and diagnosing alzheimer's diseases |
| JP2011242217A (en) * | 2010-05-17 | 2011-12-01 | Japan Health Science Foundation | Diagnostic marker of alzheimer's disease, screening method of drug for prevention and treatment of alzheimer's disease, and diagnostic method of alzheimer's disease |
| CN112336861A (en) * | 2019-08-06 | 2021-02-09 | 上海绿谷制药有限公司 | Methods of treating alzheimer's disease by modulating amino acid levels |
| CN112336859A (en) * | 2019-08-06 | 2021-02-09 | 上海绿谷制药有限公司 | Methods of treating alzheimer's disease by inhibiting amino acid uptake by T cells |
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