WO2024109767A1 - 基于粪便代谢物的阿尔兹海默症标志物及其应用 - Google Patents
基于粪便代谢物的阿尔兹海默症标志物及其应用 Download PDFInfo
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- alzheimer
- disease
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- 208000024827 Alzheimer disease Diseases 0.000 title claims abstract description 75
- 239000002207 metabolite Substances 0.000 title abstract description 17
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Definitions
- the present invention relates to the field of biotechnology, and in particular to an Alzheimer's disease marker based on fecal metabolites and an application thereof.
- AD Alzheimer's disease
- senile dementia is a progressive degenerative disease of the central nervous system that occurs in old age.
- AD is characterized by progressive memory impairment, cognitive decline, and loss of daily living ability, accompanied by neuropsychiatric symptoms such as personality changes, which seriously affect the patient's life and social life. It has become a major public health issue affecting the world.
- society many factors such as the accelerated pace of life, increased work pressure, irregular diet and work and rest have led to the younger and younger population of patients with Alzheimer's disease.
- dementia occurred more after the age of 65, but now there are many people in their 50s or even 40s suffering from the disease.
- the pathogenesis of Alzheimer's disease has not been fully clarified, and its early symptoms are relatively hidden, patients are easily missed or misdiagnosed.
- the screening of Alzheimer's disease is mainly carried out through neuropsychological scales, imaging examinations and biochemical index examination results.
- different detection methods have their limitations.
- memory scales can be used to evaluate clinical symptoms, but the sensitivity and specificity of diagnosis are low.
- positron emission tomography has the advantages of being non-invasive, in vivo, and real-time, but the detection cost is relatively high.
- biomarkers are biochemical indicators that can mark changes or possible changes in the structure or function of systems, organs, tissues, cells and subcellular structures, in order to understand the current biological state of the body, thereby providing a reference for the prevention, early diagnosis and treatment of Alzheimer's disease.
- biomarkers are beta-amyloid protein and phosphorylated tau protein in cerebrospinal fluid, but the collection of cerebrospinal fluid is invasive, which limits its clinical application.
- studying the relationship between intestinal flora metabolic homeostasis and the onset of Alzheimer's disease and screening for early diagnostic biomarkers of Alzheimer's disease based on fecal metabolites is expected to improve the accuracy of Alzheimer's disease diagnosis and help with early warning of the disease, pathological typing, and predictive evaluation of the development stage.
- finding potential biomarkers with high sensitivity and accuracy based on fecal metabolites is still a difficult task.
- the purpose of the present invention is to overcome the shortcomings of the prior art and solve the problem of low sensitivity and accuracy of the existing Alzheimer's disease biomarkers based on fecal metabolites mentioned in the above background technology.
- the present invention provides the following technical solutions:
- a biomarker for Alzheimer's disease comprising any one or more of spermidine, vitamin B5, glycylproline, vanillylmandelic acid, phenylacetylglycine, trehalose, tartaric acid, maleic acid, quinolinic acid, guanosine monophosphate, and p-hydroxyphenylpropionic acid.
- the Alzheimer's disease biomarker is a combination of any one of tartaric acid, vitamin B5, trehalose, quinolinic acid and glycylproline, maleic acid, spermidine, p-hydroxyphenylpropionic acid, and vanillylmandelic acid.
- the second aspect of the present invention provides a kit, comprising a detection tool and instructions, wherein the instructions record the process and indicators for diagnosing a subject, and is characterized in that the detection tool consists of a tool for measuring the Alzheimer's disease biomarker.
- the instructions describe the following diagnostic process: S1, determining the level of the Alzheimer's disease biomarker in a biological sample from a subject; and S2, based on the level of the Alzheimer's disease biomarker, assisting in determining or diagnosing the presence of Alzheimer's disease or the risk of developing Alzheimer's disease with high specificity.
- step S2 comprises comparing the level of the biomarker in the biological sample with a reference level of the biomarker, wherein the reference level is an average level obtained from a population that does not suffer from Alzheimer's disease.
- any one or more of the following is selected to indicate the presence of Alzheimer's disease or the risk of developing Alzheimer's disease: an increase in spermidine, a decrease in vitamin B5, an increase in glycylproline, an increase in vanillylmandelic acid, a decrease in phenylacetylglycine, a decrease in trehalose, an increase in tartaric acid, an increase in maleic acid, a decrease in quinolinic acid, a decrease in guanosine monophosphate, and an increase in parahydroxyphenylpropionic acid.
- the instructions record using the mass spectrum peak height value and/or mass spectrum peak area value of the Alzheimer's disease biomarker as a diagnostic indicator.
- the biological sample is selected from feces.
- the third aspect of the present invention provides an Alzheimer's disease biomarker for use in preparing a reagent or kit for assisting in diagnosing Alzheimer's disease in a subject and/or assisting in determining the risk of developing Alzheimer's disease in a subject, and for use in screening drugs for treating or preventing Alzheimer's disease.
- the beneficial effect of the present invention is that the Alzheimer's disease biomarker provided by this scheme can be used to assist in the diagnosis of Alzheimer's disease symptoms, has the characteristics of high detection accuracy, convenience, speed, safety and non-invasiveness, and has important clinical guiding significance for assisting the diagnosis of Alzheimer's disease related indicators.
- Figure 1 is a graph showing glycylproline content
- Figure 2 is a comparison chart of mean values of glycylproline content
- Fig. 3 is a graph showing the content of maleic acid
- FIG4 is a comparison chart of the mean values of maleic acid content
- Fig. 5 is a graph showing spermidine content
- FIG6 is a comparison diagram of the mean spermidine content
- Fig. 7 is a graph showing the p-hydroxyphenylpropionic acid content
- FIG8 is a comparison chart of the mean values of p-hydroxyphenylpropionic acid content
- Fig. 9 is a graph showing the content of vanillylmandelic acid
- Figure 10 is a comparison chart of the mean values of vanillylmandelic acid content
- Figure 11 is a graph showing tartaric acid content
- Figure 12 is a comparison chart of mean tartaric acid content
- Figure 13 is a graph showing the content of vitamin B5
- Figure 14 is a comparison chart of the mean values of vitamin B5 content
- Figure 15 is a graph showing trehalose content
- FIG16 is a comparison chart of the mean trehalose content
- Figure 17 is a graph showing quinolinic acid content
- Figure 18 is a comparison chart of the mean values of quinolinic acid content
- FIG19 is a graph showing phenylacetylglycine content
- Figure 20 is a comparison chart of the mean values of phenylacetylglycine content
- FIG21 is a graph showing guanosine monophosphate content
- FIG. 22 is a comparison chart of the mean values of guanosine monophosphate content.
- AB 5500/6500 Q-trap mass spectrometer was purchased from AB SCIEX.
- Agilent 1290 Infinity LC ultrahigh pressure liquid chromatograph was purchased from Agilent.
- Low temperature high speed centrifuge 5430R was purchased from Eppendorf.
- Chromatographic columns were purchased from Waters, and there were two types: ACQUITY UPLC BEH Amide 1.7 ⁇ m, 2.1mm ⁇ 100mm column; ACQUITY UPLC BEH C18 1.7 ⁇ m, 2.1mm ⁇ 100mm column.
- Acetonitrile was purchased from Merck, and the product number was 1499230-935.
- Ammonium acetate was purchased from Sigma, and the product number was 73594.
- Methanol was purchased from Fisher, and the product number was A456-4.
- Ammonia was purchased from Sigma, and the product number was 221228.
- Ammonium formate was purchased from Sigma, and the product number was 70221.
- Formic acid was purchased from Sigma, product number 00940.
- Isotope standards were purchased from Cambridge Isotope Laboratories.
- the sensitivity (also known as the true positive rate) described in the present invention refers to the proportion of samples that are actually positive and are judged as positive, that is, the ability to correctly judge cases that are actually sick as sick.
- Specificity (also known as the true negative rate) refers to the proportion of samples that are actually negative and are judged as negative, that is, the ability to correctly judge cases that are actually not sick.
- Accuracy also known as efficiency is expressed as the percentage of the total number of true positives and true negatives to the total number of subjects.
- the present invention provides a highly sensitive and accurate biomarker for Alzheimer's disease based on fecal metabolites.
- the combination contains 11 fecal metabolite markers, and the mass spectrum peak intensity values are used as detection indicators, which can be used to assist in the diagnosis of Alzheimer's symptoms. Because it has the characteristics of high detection accuracy, convenience, speed, safety and non-invasiveness, it has important clinical guidance significance for assisting the diagnosis of Alzheimer's disease.
- the present invention provides a kit, including a detection tool and instructions, wherein the detection tool is composed of a tool for determining Alzheimer's disease biomarkers, and the instructions record the process and indicators for diagnosing a subject.
- the diagnostic process includes: determining the level of Alzheimer's disease biomarkers in feces from the subject; based on the level of Alzheimer's disease biomarkers, assisting in determining or diagnosing the presence of Alzheimer's disease or the risk of developing Alzheimer's disease with high specificity.
- Example 1 Sample extraction and pretreatment
- mice The 9-month-old mice were divided into two groups, one was the feces group of male Alzheimer's disease model mice, namely the Fecal Transgenic (FTG) group, with a total of 10 mice; the other was the WT wild-type feces control group, namely the Fecal Wild Type (FWT) group, with a total of 9 mice.
- FGS Fecal Transgenic
- WT Fecal Wild Type
- Example 2 During mass spectrometry analysis, 100 ⁇ L of acetonitrile aqueous solution was added to the pretreated sample obtained in Example 1 for reconstitution, wherein the volume ratio of acetonitrile to water was 1:1. The sample was then vortexed and centrifuged at 14,000 ⁇ g and 4°C for 15 min, and the supernatant was sampled for analysis.
- the qualitative and quantitative information of fecal metabolites is based on targeted metabolomics analysis technology, using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC Q-TRAP/MS) for detection.
- UHPLC Q-TRAP/MS ultra-high performance liquid chromatography-triple quadrupole mass spectrometry
- This technology has high selectivity and high sensitivity, and uses targeted sample preparation and chromatographic separation methods to qualitatively and quantitatively analyze more than 300 common intestinal flora metabolites.
- the column temperature of C18 chromatographic column was 40°C, the flow rate was 0.4mL/min, and the injection volume was 2 ⁇ L; the mobile phase A was water, 5mM ammonium acetate and 0.2% ammonia water, and the mobile phase B was 99.5% acetonitrile and 0.5% ammonia water; the elution gradient was: 0-5min B phase linearly changed from 5% to 60%, 5-11min B phase linearly changed from 60% to 100%, 11-13min B phase maintained at 100%, 13-13.1min B phase linearly changed from 100% to 5%, 13.1-16min B phase maintained at 5%; the samples were placed in the automatic sampler at 4°C during the entire analysis process.
- samples were analyzed continuously in random order.
- QC samples were inserted into the sample queue, that is, samples obtained by mixing all samples to be tested with the same volume and using the same pretreatment method as the samples to be tested, which were used to monitor and evaluate the stability of the system and the reliability of experimental data.
- Mass spectrometry conditions The mass spectrometer was an AB 6500 QTRAP system from AB SCIEX. Electrospray ionization (ESI) was used as the ionization mode, and the parameters were set as follows: sheath gas temperature 350°C; drying gas temperature 350°C; sheath gas flow rate 11 L/min; drying gas flow rate 10 L/min; capillary voltage 4000 V in positive ion mode and -3500 V in negative ion mode; nozzle voltage 500 V; nebulization pressure 30 psi; and mass spectrometry multiple reaction monitoring (MRM) was used.
- ESI Electrospray ionization
- the intergroup difference analysis of the two groups of mice showed that the levels of the following metabolites in the AD group mice were significantly different from those in the control group, indicating that these metabolites play an important role in distinguishing the Alzheimer's disease model: Spermidine, Vitamin B5, Glycyl-L-proline, Vanillylmandelic acid, Phenylacetylglycine, Trehalose, Tartaric acid, Maleic acid, Quinolinic acid, Guanosine monophosphate, and P-Hydroxyl phenylpropanol.
- Figures 1-10 show the contents of glycylproline, maleic acid, spermidine, p-hydroxyphenylpropionic acid, and vanillylmandelic acid. It can be seen that the contents of these two biomarkers in the feces of mice with Alzheimer's disease show an overall increasing trend.
- Figures 11-18 show the contents of tartaric acid, vitamin B5, trehalose, and quinolinic acid, respectively. The reduction in the contents of these biomarkers indicates that the subject has Alzheimer's disease or is at risk of developing Alzheimer's disease.
- Figures 19-22 show the contents of phenylacetylglycine and guanosine monophosphate, respectively. The average contents of these biomarkers in mice with Alzheimer's disease show a decreasing trend relative to mice without the disease, indicating that the subjects are at risk of developing Alzheimer's disease.
- spermidine belongs to amines
- vitamin B5 belongs to vitamins
- glycylproline belongs to amino acids
- vanillylmandelic acid and phenylacetylglycine belong to benzene ring compounds
- trehalose and tartaric acid belong to carbohydrates
- maleic acid belongs to dicarboxylic acids
- quinolinic acid and guanosine monophosphate belong to purine nucleotides
- p-hydroxyphenylpropionic acid belongs to phenols.
- AD patients have abnormalities in multiple metabolic pathways such as amines, vitamins, amino acids, benzene ring compounds, carbohydrates, dicarboxylic acids, purine nucleotides, and phenols, and changes in fecal metabolite levels may reflect abnormalities in related metabolic pathways in the brains of Alzheimer's patients, which is of great significance for early clinical diagnosis.
- the present invention uses the mass spectrometry peak intensity values of 11 fecal metabolic markers as detection targets or evaluation indicators, which can be used to assist in the diagnosis of Alzheimer's disease symptoms. It has the characteristics of high detection accuracy, convenience, speed, safety and non-invasiveness, and has important clinical guiding significance for assisting the diagnosis of Alzheimer's disease-related indicators.
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Abstract
本发明涉及生物技术领域，具体涉及一种基于粪便代谢物的阿尔兹海默症标志物及其应用。本方案提供的生物标志物具有高灵敏度、高准确度的特点，将其质谱峰强度值作为检测指标，能够以检测精确度高、方便快捷以及安全无创的方式为阿尔兹海默症的临床早期辅助诊断提供重要参考。
Description
本发明涉及生物技术领域，具体涉及一种基于粪便代谢物的阿尔兹海默症标志物及其应用。
阿尔兹海默症（Alzheimer disease，AD），又称老年痴呆症，是一种发生于老年期的进行性发展的中枢神经系统退行性变性疾病。阿尔兹海默症以渐进性记忆障碍、认知功能下降以及日常生活能力丧失为特征，并伴随有人格改变等神经精神症状，严重影响了患者的生活与社交，目前已成为影响全球的重大公共健康问题。随着社会发展，生活节奏加快、工作压力变大、饮食与作息不规律等诸多因素导致阿尔兹海默症的患病人群日趋年轻化，原本痴呆症多于65岁后发病，现在却有不少50多岁甚至40多岁的人患病。此外，由于阿尔兹海默症的发病机制尚未完全明确，加上其早期症状比较隐秘，导致患者容易被漏诊或错诊。
现阶段对阿尔兹海默症的筛查主要通过神经心理学量表、影像学检查以及生化指标检查结果。然而，不同检测方式均有其局限性。比如，记忆量表能够用于评估临床症状，但诊断的灵敏度和特异性较低。又比如，正电子发射型计算机断层显像具有无创、在体、实时等优势，但检测成本较高。
为此，人们研究了多样的生物标志物，即可以标记系统、器官、组织、细胞及亚细胞结构或功能的改变或可能发生的改变的生化指标，以获知机体当前所处生物学状态，从而为阿尔兹海默症的预防、早期诊断和治疗提供参考。目前被广泛接受的生物标志物有脑脊液中的β-淀粉样蛋白和磷酸化tau蛋白，但脑脊液采集具有创伤性，限制了其临床应用。
进一步地，有研究通过获取方便的粪便了解肠道菌群情况，希望能以快速、非侵入且成本低廉的策略完成阿尔兹海默症的早期诊断。在最近的研究中，人们发现多种神经精神疾病，如帕金森、抑郁症、自闭症等均与肠道菌群失衡有关，并且80%以上阿尔兹海默症患者存在肠道菌群失衡的现象，这提示肠道菌群稳态与阿尔兹海默症等神经退行性疾病的发病进程存在密切关联。
有数据显示，阿尔兹海默症患者肠道菌群组成与健康同龄人不同，使得宿主和肠道菌群在代谢食物物质的过程中产生了大量不同的代谢物，这种肠道菌群多样性和丰度的改变也会对机体中小分子代谢物的种类和浓度产生重要影响。此外，各种代谢途径的紊乱可能会反向介导阿尔兹海默症的病理发生和发展。有研究表明，机体发生代谢紊乱可能会导致阿尔兹海默症中菌群的失衡，同时，机体外周代谢的改变又可能通过血液循环进一步加大中枢神经系统代谢的紊乱。与此同时，随着衰老及年龄增长，肠黏膜屏障的通透性增加，导致一些机会致病菌和其代谢产物穿过屏障入侵内部组织器官，诱发了慢性肠道及全身炎症，这一现象也为阿尔兹海默症患病风险与年龄的高度正相关性提供了一种合理的解释。
因此，研究肠道菌群代谢稳态与阿尔兹海默症发病的关系，基于粪便代谢物筛选阿尔兹海默症早期诊断生物标志物，预期可以提高阿尔兹海默症诊断的准确性，有助于疾病的早期预警、病理分型以及发展阶段的预测评估等。然而，基于粪便代谢物，寻找具有高灵敏度、高准确度的潜在生物标志物仍是一项困难的工作。
本发明的目的在于克服现有技术不足，解决上述背景技术中提到的现有基于粪便代谢物的阿尔兹海默症生物标志物灵敏度和准确度较低问题。
为实现上述目的，本发明提供以下技术方案：
本发明的第一方面，提供一种阿尔兹海默症生物标志物，包括亚精胺、维生素B5、甘氨酰脯氨酸、香草扁桃酸、苯乙酰甘氨酸、海藻糖、酒石酸、马来酸、喹啉酸、鸟苷一磷酸、对羟基苯丙酸中的任意一种或多种。
优选地，所述阿尔兹海默症生物标志物为酒石酸、维生素B5、海藻糖、喹啉酸中任意一种和甘氨酰脯氨酸、马来酸、亚精胺、对羟基苯丙酸、香草扁桃酸的组合。
本发明的第二方面，提供一种试剂盒，包括检测工具和说明书，所述说明书记载了诊断受试者的流程和指标，其特征在于，所述检测工具由用于测定所述阿尔兹海默症生物标志物的工具组成。
在一些实施例中，所述说明书记载了以下诊断流程：S1、测定来自受试者的生物样品中所述阿尔兹海默症生物标志物的水平；和S2、基于所述阿尔兹海默症生物标志物的水平，以高的特异性辅助确定或诊断阿尔兹海默症的存在或发生阿尔兹海默症的风险。
在一些实施例中，所述步骤S2包括将所述生物样品中所述生物标志物的水平与所述生物标志物的参比水平进行比较，其中所述参比水平是从未患有阿尔兹海默症的群体获得的平均水平。
在一些实施例中，所述步骤S2中，选自以下任意一种或多种指示阿尔兹海默症的存在或发生阿尔兹海默症的风险：亚精胺的增多、维生素B5的减少、甘氨酰脯氨酸的增多、香草扁桃酸的增多、苯乙酰甘氨酸的减少、海藻糖的减少、酒石酸的增多、马来酸的增多、喹啉酸的减少、鸟苷一磷酸的减少、对羟基苯丙酸的增多。
在一些实施例中，所述说明书记载了以所述阿尔兹海默症生物标志物的质谱峰高度值和/或质谱峰面积值作为诊断指标。
在一些实施例中，所述生物样品选自粪便。
本发明的第三方面，提供一种阿尔兹海默症生物标志物在制备辅助诊断受试者中阿尔兹海默症和/或辅助确定受试者中阿尔兹海默症发生风险的试剂或试剂盒中的应用和筛选治疗或预防阿尔兹海默症药物中的应用。
与现有技术相比，本发明的有益效果是本方案所提供的阿尔兹海默症生物标志物，能够用于阿尔兹海默症的症状辅助判断，具有检测精确度高、方便快捷以及安全无创的特点，对辅助诊断出阿尔兹海默症相关指标具有重要的临床指导意义。
图1为甘氨酰脯氨酸含量图；
图2为甘氨酰脯氨酸含量均值对比图；
图3为马来酸含量图；
图4为马来酸含量均值对比图；
图5为亚精胺含量图；
图6为亚精胺含量均值对比图；
图7为对羟基苯丙酸含量图；
图8为对羟基苯丙酸含量均值对比图；
图9为香草扁桃酸含量图；
图10为香草扁桃酸含量均值对比图；
图11为酒石酸含量图；
图12为酒石酸含量均值对比图；
图13为维生素B5含量图；
图14为维生素B5含量均值对比图；
图15为海藻糖含量图；
图16为海藻糖含量均值对比图；
图17为喹啉酸含量图；
图18为喹啉酸含量均值对比图；
图19为苯乙酰甘氨酸含量图；
图20为苯乙酰甘氨酸含量均值对比图；
图21为鸟苷一磷酸含量图；
图22为鸟苷一磷酸含量均值对比图。
下面将结合具体实施方式对本专利的技术方案作进一步详细地说明，应该指出，以下详细说明都是示例性的，旨在对本申请提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
本发明试验及仪器来源：AB 5500/6500 Q-trap质谱仪购于AB SCIEX公司。Agilent 1290 Infinity LC超高压液相色谱仪购于Agilent公司。低温高速离心机5430R购于Eppendorf公司。色谱柱购于Waters公司，共有两种：ACQUITY UPLC BEH Amide 1.7μm，2.1mm×100mm column；ACQUITY UPLC BEH C18 1.7μm，2.1mm×100mm column。乙腈购于Merck公司，产品编号为1499230-935。乙酸铵购于Sigma公司，产品编号为73594。甲醇购于Fisher公司，产品编号为A456-4。氨水购于Sigma公司，产品编号为221228。甲酸铵购于Sigma公司，产品编号为70221。甲酸购于Sigma公司，产品编号为00940。同位素标准品购于Cambridge Isotope Laboratories公司。
需要说明的是，本发明中所述灵敏度（Sensitivity，也称真阳性率）是指在实际为阳性的样本中将其判断为阳性的比例，即能将实际患病的病例正确地判断为患病的能力。特异度（Specificity，也称真阴性率）是指在实际为阴性的样本中将其判断为阴性的比例，即能正确判断实际未患病的病例的能力。准确度（Accuracy，也称效率）则用真阳性与真阴性总数占受试者总数的百分率表示。
本发明提供一种基于粪便代谢物的高灵敏度、高准确度的阿尔兹海默症生物标志物。该组合共含11种粪便代谢标志物，以其质谱峰强度值作为检测指标，能够用于阿尔兹海默症的症状辅助判断。因其具有检测精确度高、方便快捷以及安全无创的特点，所以对辅助诊断阿尔兹海默症具有重要的临床指导意义。
基于上述生物标志物，本发明提供一种试剂盒，包括检测工具和说明书，其中检测工具由用于测定阿尔兹海默症生物标志物的工具组成，说明书记载了诊断受试者的流程和指标。诊断流程包括：测定来自受试者的粪便中阿尔兹海默症生物标志物的水平；基于阿尔兹海默症生物标志物的水平，以高的特异性辅助确定或诊断阿尔兹海默症的存在或发生阿尔兹海默症的风险。
实施例1：样品提取和预处理
将9月龄的小鼠分为两组，一组为阿尔兹海默症雄性模型小鼠粪便组，即Fecal Transgenic（FTG）组，共10只；另一组为WT野生型粪便对照组，即Fecal Wild Type（FWT）组，共9只。
取适量从阿尔兹海默症患病个体和正常健康对照供体中采集的粪便样品，加入预冷的甲醇/乙腈/水溶液，其中甲醇、乙腈、水的体积比为2:2:1。之后进行涡旋混合并于低温下超声30min，而后在-20℃静置10 min，于14,000×g、4℃的条件下离心20min，取上清液进行真空干燥，得到预处理样品。
实施例2：LC-MS/MS分析
（1）质谱分析样品的制备。
质谱分析时，向实施例1得到的预处理样品中加入100μL乙腈水溶液复溶，其中乙腈和水的体积比为1:1。之后经涡旋混合在14,000×g、4℃的条件下离心15 min，并取上清液进样分析。
（2）LC-MS/MS条件。
粪便代谢物的定性定量信息基于靶向代谢组学分析技术，采用超高效液相色谱-三重四级杆质谱联用仪（UHPLC Q-TRAP/MS）进行检测。该技术具有高选择能力和高灵敏度，使用针对性开发的样品制备及色谱分离方法，能够定性和定量分析三百多种常见的肠道菌群代谢产物。
色谱条件。采用Agilent 1290 Infinity LC型超高效液相色谱系统搭配HILIC和C18色谱柱分离样品。HILIC色谱柱柱温35℃，流速0.3mL/min，进样量2μL；流动相A为90%水、2 mM甲酸铵和10%乙腈，流动相B为甲醇和0.4%甲酸；洗脱梯度：0-1.0min为85% B相，1.0-3.0min B相从85%线性变化至80%，3.0-4.0min为80% B相，4.0-6.0min B相从80%线性变化至70%，6.0-10.0min B相从70%线性变化至50%，10-12.5min B相维持在50%，12.5-12.6min B相从50%线性变化至85%，12.6-18min B相维持在85%。C18色谱柱柱温40℃，流速0.4mL/min，进样量 2μL；流动相A为水、5mM乙酸铵和0.2%氨水，流动相B为99.5%乙腈和0.5%氨水；洗脱梯度：0-5min B相从5%线性变化至60%，5-11min B相从60%线性变化至100%，11-13min B相维持在100%，13-13.1min B相从100%线性变化至5%，13.1-16min B相维持在5%；整个分析过程中样品置于4℃自动进样器中。为避免仪器检测信号波动带来的影响，采用随机顺序进行样本的连续分析。样本队列中插入QC样品，即混合相同体积的所有待检测样本后按照与待测样本相同的前处理方法得到的样本，用于监测和评价系统的稳定性及实验数据的可靠性。
质谱条件。质谱仪为AB SCIEX公司的AB 6500 QTRAP系统。采用电喷雾离子源（ESI）作为离子化方式，参数设置如下：鞘气温度350℃；干燥气温度350℃；鞘气流速11L/min；干燥气流速10 L/min；毛细管电压正离子模式下为4000V，负离子模式下为-3500V；喷嘴电压500 V；雾化压力30 psi；采用质谱多反应监测（Multiple Reaction Monitoring，MRM）。
数据分析。使用MultiQuant或Analyst软件对MRM原始数据进行峰提取，得到各物质的峰面积和内标峰面积的比值，并根据标准曲线计算含量。
（3）结果分析。
如图1-13所示，对两组小鼠进行组间差异对比分析，可以发现AD组小鼠如下几种代谢物水平与对照组相比有显著差异，说明了该部分代谢物对于阿尔兹海默症模型的区分有重要作用：亚精胺（Spermidine）、维生素B5（Vitamin B5）、甘氨酰脯氨酸（Glycyl-L-proline）、香草扁桃酸（Vanillylmandelic acid）、苯乙酰甘氨酸（Phenylacetylglycine）、海藻糖（Trehalose）、酒石酸（Tartaric acid）、马来酸（Maleic acid）、喹啉酸（Quinolinic acid）、鸟苷一磷酸（Guanosine monophosphate）、对羟基苯丙酸（P-Hydroxyl phenylpropanol）。
具体来讲，图1-10展示了甘氨酰脯氨酸、马来酸、亚精胺、对羟基苯丙酸、香草扁桃酸的含量情况，可以看出患阿尔兹海默症小鼠粪便中的这两种生物标志物的含量均呈现整体增加的趋势。图11-18分别为酒石酸、维生素B5、海藻糖和喹啉酸含量情况，这部分生物标志物含量减少指示受试者存在阿尔兹海默症或具有发生阿尔兹海默症的风险。图19-22分别为苯乙酰甘氨酸、鸟苷一磷酸含量情况，患阿尔兹海默症小鼠的此部分生物标志物含量平均值相对于未患疾病小鼠有减少趋势，提示受试者存在患阿尔兹海默症的风险。
进一步地，对以上代谢物进行分类分析，发现亚精胺属于胺类，维生素B5属于维生素，甘氨酰脯氨酸属于氨基酸，香草基扁桃酸和苯乙酰甘氨酸属于苯环型化合物，海藻糖和酒石酸属于碳水化合物，马来酸属于二羧酸，喹啉酸和鸟苷一磷酸属于嘌呤核苷酸，对羟基苯丙酸属于酚类。这些结果提示AD患者体内存在胺类、维生素、氨基酸、苯环型化合物、碳水化合物、二羧酸、嘌呤核苷酸、酚类等多种代谢通路的异常，而粪便代谢物水平变化可能反映阿尔兹海默症患者脑中相关代谢通路的异常，对临床早期诊断具有重要意义。
本发明以11种粪便代谢标志物的质谱峰强度值作为检测靶点或评估指标，能够用于阿尔兹海默症的症状辅助判断，具有检测精确度高、方便快捷以及安全无创的特点，对辅助诊断出阿尔兹海默症相关指标具有重要的临床指导意义。
以上所述仅是本发明的一些实施方式。对于本领域技术人员来说，在不脱离本发明创造构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。
Claims (10)
- 一种阿尔兹海默症生物标志物，其特征在于，所述生物标志物包括：亚精胺、维生素B5、甘氨酰脯氨酸、香草扁桃酸、苯乙酰甘氨酸、海藻糖、酒石酸、马来酸、喹啉酸、鸟苷一磷酸、对羟基苯丙酸中的任意一种或多种。
- 根据权利要求1所述阿尔兹海默症生物标志物，其特征在于，所述生物标志物为酒石酸、维生素B5、海藻糖、喹啉酸中任意一种和甘氨酰脯氨酸、马来酸、亚精胺、对羟基苯丙酸、香草扁桃酸的组合。
- 一种试剂盒，包括检测工具和说明书，所述说明书记载了诊断受试者的流程和指标，其特征在于，所述检测工具由用于测定权利要求1或2所述阿尔兹海默症生物标志物的工具组成。
- 根据权利要求2所述的试剂盒，其特征在于，所述说明书记载了以下诊断流程：S1、测定来自受试者的生物样品中所述阿尔兹海默症生物标志物的水平；和S2、基于所述阿尔兹海默症生物标志物的水平，以高的特异性辅助确定或诊断阿尔兹海默症的存在或发生阿尔兹海默症的风险。
- 根据权利要求3所述的试剂盒，其特征在于，所述步骤S2包括将所述生物样品中所述生物标志物的水平与所述生物标志物的参比水平进行比较，其中所述参比水平是从未患有阿尔兹海默症的群体获得的平均水平。
- 根据权利要求4所述的试剂盒，其特征在于，所述步骤S2中，选自以下任意一种或多种指示阿尔兹海默症的存在或发生阿尔兹海默症的风险：亚精胺的增多、维生素B5的减少、甘氨酰脯氨酸的增多、香草扁桃酸的增多、苯乙酰甘氨酸的减少、海藻糖的减少、酒石酸的增多、马来酸的增多、喹啉酸的减少、鸟苷一磷酸的减少、对羟基苯丙酸的增多。
- 根据权利要求2所述的试剂盒，其特征在于，所述说明书记载了以所述阿尔兹海默症生物标志物的质谱峰高度值和/或质谱峰面积值作为诊断指标。
- 根据权利要求2所述的试剂盒，其特征在于，所述生物样品选自粪便。
- 权利要求1或2所述阿尔兹海默症生物标志物在制备辅助诊断受试者中阿尔兹海默症和/或辅助确定受试者中阿尔兹海默症发生风险的试剂或试剂盒中的应用。
- 权利要求1或2所述阿尔兹海默症生物标志物在筛选治疗或预防阿尔兹海默症药物中的应用。
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