WO2024109768A1 - Alzheimer's disease marker based on blood metabolite and use thereof - Google Patents
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Abstract
The present invention relates to the field of biotechnology, and in particular to an Alzheimer's disease marker based on a blood metabolite and the use thereof. A biomarker provided by the present solution has the characteristics of high sensitivity and high accuracy, and can provide an important reference for the clinical early auxiliary diagnosis of Alzheimer's disease in a highly accurate, convenient, fast and safe and non-invasive manner in detection by means of using a peak intensity value of a mass spectrum thereof as a detection index.
Description
本发明涉及生物技术领域,具体涉及一种基于血液代谢物的阿尔茨海默症标志物及其应用。The present invention relates to the field of biotechnology, and in particular to an Alzheimer's disease marker based on blood metabolites and an application thereof.
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。Alzheimer's disease (AD), also known as 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. With the development of 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. Originally, dementia occurred more after the age of 65, but now there are many people in their 50s or even 40s suffering from the disease. In addition, because the pathogenesis of Alzheimer's disease has not been fully clarified, and its early symptoms are relatively hidden, patients are easily missed or misdiagnosed.
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。At present, the screening of Alzheimer's disease is mainly carried out through neuropsychological scales, imaging examinations and biochemical index examination results. However, different detection methods have their limitations. For example, memory scales can be used to evaluate clinical symptoms, but the sensitivity and specificity of diagnosis are low. For another example, positron emission tomography has the advantages of being non-invasive, in vivo, and real-time, but the detection cost is relatively high.
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,研究转向了获取方便、侵入性小的血液,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。To this end, people have studied a variety of biomarkers, that is, 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. Currently, the widely accepted biomarkers are beta-amyloid protein and phosphorylated tau protein in cerebrospinal fluid, but the collection of cerebrospinal fluid is traumatic, which limits its clinical application. Further, the research turned to blood, which is easy to obtain and less invasive, hoping to complete the early diagnosis of Alzheimer's disease with a rapid, non-invasive and low-cost strategy.
近年来,基因组学、转录组学、蛋白组学及代谢组学等高通量组学技术快速发展,使得新型生物标志物的研发更进一步。其中,代谢组学利用磁共振波谱和质谱等技术监测代谢产物图谱的动态变化,有效筛选了疾病相关生物标志物,因而在阐明阿尔兹海默症的分子致病机制及其导致的病理生理变化上展现出了广阔的应用前景。血液中含有蛋白质、多肽、核酸、脂质和其他代谢物,能够帮助阐明阿尔兹海默症的复杂性和异质性。因此,基于血液中的代谢物筛选阿尔兹海默症早期诊断生物标志物,预期可以提高疾病诊断的准确性,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,基于血液代谢物,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。In recent years, the rapid development of high-throughput omics technologies such as genomics, transcriptomics, proteomics and metabolomics has made the research and development of new biomarkers a step further. Among them, metabolomics uses technologies such as magnetic resonance spectroscopy and mass spectrometry to monitor the dynamic changes of metabolite profiles and effectively screen disease-related biomarkers, thus showing broad application prospects in clarifying the molecular pathogenic mechanism of Alzheimer's disease and the pathophysiological changes caused by it. Blood contains proteins, peptides, nucleic acids, lipids and other metabolites, which can help clarify the complexity and heterogeneity of Alzheimer's disease. Therefore, screening for early diagnostic biomarkers of Alzheimer's disease based on metabolites in the blood is expected to improve the accuracy of disease diagnosis, help early warning of the disease, pathological typing, and predictive evaluation of the development stage. However, based on blood metabolites, it is still a difficult task to find potential biomarkers with high sensitivity and high accuracy.
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血液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。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 blood metabolites mentioned in the above background technology.
为实现上述目的,本发明提供以下技术方案:To achieve the above object, the present invention provides the following technical solutions:
本发明的第一方面,提供一种阿尔兹海默症生物标志物,包括三甲胺-N-氧化物、莽草酸、3-氨基水杨酸、马尿酸、N-乙酰色氨酸、D-葡萄糖、羟钴胺、核黄素-5’-磷酸、羟基丙酮酸、羟基乙酸、3-羟基-2-丁酮、甜菜碱、黄尿酸中的任意一种或多种。In a first aspect of the present invention, a biomarker for Alzheimer's disease is provided, comprising any one or more of trimethylamine-N-oxide, shikimic acid, 3-aminosalicylic acid, hippuric acid, N-acetyltryptophan, D-glucose, hydroxycobalamin, riboflavin-5'-phosphate, hydroxypyruvic acid, glycolic acid, 3-hydroxy-2-butanone, betaine, and xanthuric acid.
优选地,所述阿尔兹海默症生物标志物为三甲胺-N-氧化物、D-葡萄糖、莽草酸和羟基丙酮酸的组合。Preferably, the Alzheimer's disease biomarker is a combination of trimethylamine-N-oxide, D-glucose, shikimic acid and hydroxypyruvic acid.
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。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.
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。In some embodiments, 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.
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。In some embodiments, 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.
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:三甲胺-N-氧化物的减少、莽草酸的减少、3-氨基水杨酸的减少、N-乙酰色氨酸的减少、D-葡萄糖的减少、核黄素-5’-磷酸的减少、羟基丙酮酸的减少、3-羟基-2-丁酮的减少、甜菜碱的减少、黄尿酸的减少、马尿酸的减少、羟钴胺的减少、羟基乙酸的减少。In some embodiments, in step S2, 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: a decrease in trimethylamine-N-oxide, a decrease in shikimic acid, a decrease in 3-aminosalicylic acid, a decrease in N-acetyltryptophan, a decrease in D-glucose, a decrease in riboflavin-5'-phosphate, a decrease in hydroxypyruvic acid, a decrease in 3-hydroxy-2-butanone, a decrease in betaine, a decrease in xanthuric acid, a decrease in hippuric acid, a decrease in hydroxycobalamin, and a decrease in glycolic acid.
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。In some embodiments, 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.
在一些实施例中,所述生物样品选自血液。In some embodiments, the biological sample is selected from blood.
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筛选治疗或预防阿尔兹海默症药物、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。The third aspect of the present invention provides an Alzheimer's disease biomarker for use in preparing a reagent or kit for screening drugs for treating or preventing Alzheimer's disease, assisting in diagnosing Alzheimer's disease in a subject, and/or assisting in determining the risk of developing Alzheimer's disease in a subject.
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。Compared with the prior art, 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.
图1为三甲胺-N-氧化物含量图;Figure 1 is a graph showing the content of trimethylamine-N-oxide;
图2为三甲胺-N-氧化物含量均值对比图;Figure 2 is a comparison chart of the mean values of trimethylamine-N-oxide content;
图3为D-葡萄糖含量图;Fig. 3 is a graph showing D-glucose content;
图4为D-葡萄糖含量均值对比图;FIG4 is a comparison diagram of mean values of D-glucose content;
图5为莽草酸含量图;Fig. 5 is a graph showing shikimic acid content;
图6为莽草酸含量均值对比图;FIG6 is a comparison chart of the mean values of shikimic acid content;
图7为羟基丙酮酸含量图;Fig. 7 is a graph showing hydroxypyruvic acid content;
图8为羟基丙酮酸含量均值对比图;FIG8 is a comparison chart of the mean values of hydroxypyruvic acid content;
图9为3-氨基水杨酸含量图;Fig. 9 is a graph showing the content of 3-aminosalicylic acid;
图10为3-氨基水杨酸含量均值对比图;Figure 10 is a comparison chart of the mean values of 3-aminosalicylic acid content;
图11为N-乙酰色氨酸含量图;Figure 11 is a graph showing N-acetyltryptophan content;
图12为N-乙酰色氨酸含量均值对比图;Figure 12 is a comparison chart of mean values of N-acetyltryptophan content;
图13为核黄素-5’-磷酸含量图;FIG13 is a graph showing riboflavin-5′-phosphate content;
图14为核黄素-5’-磷酸含量均值对比图;FIG14 is a comparison chart of the mean values of riboflavin-5′-phosphate content;
图15为3-羟基-2-丁酮含量图;Figure 15 is a graph showing 3-hydroxy-2-butanone content;
图16为3-羟基-2-丁酮含量均值对比图;Figure 16 is a comparison chart of the mean values of 3-hydroxy-2-butanone content;
图17为甜菜碱含量图;Figure 17 is a graph showing betaine content;
图18为甜菜碱含量均值对比图;Figure 18 is a comparison chart of mean betaine content;
图19为黄尿酸含量图;Figure 19 is a graph showing xanthuric acid content;
图20为黄尿酸含量均值对比图;Figure 20 is a comparison chart of the mean values of xanthuric acid content;
图21为马尿酸含量图;Figure 21 is a graph showing hippuric acid content;
图22为马尿酸含量均值对比图;Figure 22 is a comparison chart of the mean values of hippuric acid content;
图23为羟钴胺含量图;FIG23 is a graph showing hydroxocobalamin content;
图24为羟钴胺含量均值对比图;FIG24 is a comparison chart of the mean values of hydroxocobalamin content;
图25为羟基乙酸含量图;Fig. 25 is a graph showing glycolic acid content;
图26为羟基乙酸含量均值对比图。FIG. 26 is a comparison chart of the mean values of glycolic acid content.
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。The technical solution of this patent will be further described in detail below in conjunction with specific implementation methods. It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by ordinary technicians in the technical field to which this application belongs.
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。The sources of the experiment and instruments of the present invention are as follows: 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 1.7μm, 2.1mm×100mm column; ACQUITY UPLC BEH C18 1.7μm, 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.
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。It should be noted that 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.
本发明提供一种基于血液代谢物的高灵敏度、高准确度的阿尔兹海默症生物标志物。该组合共含13种血液代谢标志物,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。The present invention provides a highly sensitive and accurate Alzheimer's disease biomarker based on blood metabolites. The combination contains 13 blood 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 disease 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.
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:测定来自受试者的血清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。Based on the above biomarkers, 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 serum from a 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.
实施例1:样品提取和预处理Example 1: Sample extraction and pretreatment
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血清组,即Serum Transgenic(STG)组,共10只;另一组为WT野生型血清对照组,即Serum Wild Type(SWT)组,共9只。The 9-month-old mice were divided into two groups, one group was the Alzheimer's disease male model mouse serum group, namely the Serum Transgenic (STG) group, with a total of 10 mice; the other group was the WT wild-type serum control group, namely the Serum Wild Type (SWT) group, with a total of 9 mice.
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血清样品,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。Take an appropriate amount of serum samples collected from individuals with Alzheimer's disease and normal healthy control donors, add pre-cooled methanol/acetonitrile/water solution, where the volume ratio of methanol, acetonitrile and water is 2:2:1. Then vortex mix and sonicate at low temperature for 30 minutes, then stand at -20℃ for 10 minutes, centrifuge at 14,000×g and 4℃ for 20 minutes, and take the supernatant for vacuum drying to obtain the pre-treated sample.
实施例2:LC-MS/MS分析Example 2: LC-MS/MS analysis
(1)质谱分析样品的制备。(1) Preparation of samples for mass spectrometry analysis.
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。During mass spectrometry analysis, 100 μL 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.
(2)LC-MS/MS条件。(2) LC-MS/MS conditions.
血液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。The qualitative and quantitative information of blood metabolites is based on targeted metabolomics analysis technology, using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC Q-TRAP/MS) for detection. 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.
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为水、100 mM乙酸铵和1.2%氨水,流动相B为乙腈;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、50mM甲酸铵和0.4%甲酸,流动相B为甲醇;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。Chromatographic conditions. Agilent 1290 Infinity LC ultra-high performance liquid chromatography system with HILIC and C18 columns were used to separate the samples. The column temperature of the HILIC column was 35°C, the flow rate was 0.3 mL/min, and the injection volume was 2 μL; the mobile phase A was water, 100 mM ammonium acetate, and 1.2% ammonia water, and the mobile phase B was acetonitrile; the elution gradient was: 0-1.0 min was 85% B phase, 1.0-3.0 min B phase changed linearly from 85% to 80%, 3.0-4.0 min was 80% B phase, 4.0-6.0 min B phase changed linearly from 80% to 70%, 6.0-10.0 min B phase changed linearly from 70% to 50%, 10-12.5 min B phase was maintained at 50%, 12.5-12.6 min B phase changed linearly from 50% to 85%, and 12.6-18 min B phase was maintained at 85%. The column temperature of C18 chromatographic column was 40℃, the flow rate was 0.4mL/min, and the injection volume was 2μL; the mobile phase A was water, 50mM ammonium formate and 0.4% formic acid, and the mobile phase B was methanol; the elution gradient was as follows: 0-5min B phase changed linearly from 5% to 60%, 5-11min B phase changed linearly from 60% to 100%, 11-13min B phase was maintained at 100%, 13-13.1min B phase changed linearly from 100% to 5%, and 13.1-16min B phase was maintained at 5%; the samples were placed in the automatic sampler at 4℃ during the entire analysis process. In order to avoid the influence of fluctuations in instrument detection signals, the 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 following 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.
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)。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.
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。Data analysis: Use MultiQuant or Analyst software to perform peak extraction on the MRM raw data, obtain the ratio of the peak area of each substance to the peak area of the internal standard, and calculate the content based on the standard curve.
(3)结果分析。(3) Result analysis
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:三甲胺-N-氧化物(Trimethylamine-N-oxide,TMAO)、莽草酸(Shikimic acid)、3-氨基水杨酸(3-Aminosalicylic acid)、马尿酸(Hippuric acid)、N-乙酰色氨酸(N-acetyltryptophan,NAT)、D-葡萄糖(D-Glucose)、羟钴胺(Hydroxocobalamin)、核黄素-5’-磷酸(Riboflavin-5’-monophosphate,FMN)、羟基丙酮酸(Hydroxypyruvic acid)、羟基乙酸(Glycolic acid)、3-羟基-2-丁酮(Acetoin)、甜菜碱(Betaine)、黄尿酸(Xanthurenate)。As shown in Figure 1-13, 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: Trimethylamine-N-oxide (TMAO), Shikimic acid, 3-Aminosalicylic acid, Hippuric acid, N-acetyltryptophan (NAT), D-glucose, Hydroxocobalamin, Riboflavin-5'-monophosphate (FMN), Hydroxypyruvic acid, Glycolic acid, Acetoin, Betaine, and Xanthurenate.
具体来讲,图1-8展示了三甲胺-N-氧化物、D-葡萄糖、莽草酸和羟基丙酮酸的含量情况,可以看出患阿尔兹海默症小鼠血清中的这四种生物标志物的含量均呈现整体减少的趋势。同时,这些生物标志物的平均值在两组间也存在较为明显的差异,提示葡萄糖代谢通路在阿尔兹海默症中可能存在异常改变。图9-26分别为3-氨基水杨酸、N-乙酰色氨酸、核黄素-5’-磷酸、3-羟基-2-丁酮、甜菜碱、黄尿酸、马尿酸、羟钴胺、羟基乙酸含量图,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降低趋势,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。Specifically, Figures 1-8 show the contents of trimethylamine-N-oxide, D-glucose, shikimic acid, and hydroxypyruvic acid. It can be seen that the contents of these four biomarkers in the serum of mice with Alzheimer's disease all show an overall decreasing trend. At the same time, there are also obvious differences in the average values of these biomarkers between the two groups, suggesting that there may be abnormal changes in the glucose metabolism pathway in Alzheimer's disease. Figures 9-26 are respectively 3-aminosalicylic acid, N-acetyltryptophan, riboflavin-5'-phosphate, 3-hydroxy-2-butanone, betaine, xanthurenic acid, hippuric acid, hydroxocobalamin, and glycolic acid content graphs. The average values of these biomarker contents in mice with Alzheimer's disease have a decreasing trend relative to mice without the disease, indicating that the subjects have Alzheimer's disease or are at risk of developing Alzheimer's disease.
进一步地,对以上代谢物进行分类分析,发现三甲胺-N-氧化物属于氨基氧化物,莽草酸属于有机酸,3-氨基水杨酸、马尿酸、N-乙酰色氨酸则属于苯环型化合物,D-葡萄糖属于碳水化合物,羟钴胺属于咕啉类,核黄素-5’-磷酸则属于黄素核苷酸,羟基丙酮酸、羟基乙酸属于羟基酸,3-羟基-2-丁酮属于有机氧化合物,甜菜碱属于嘧啶,黄尿酸属于喹啉衍生物。这些结果提示阿尔兹海默症患者体内存在氨基氧化物类、有机酸类、苯环型化合物类、碳水化合物类、咕啉类、核苷酸类、羟基酸类、有机氧化物类、嘧啶类以及喹啉衍生物类等多种代谢通路的异常,而血液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。Further, the above metabolites were classified and analyzed, and it was found that trimethylamine-N-oxide belongs to amino oxides, shikimic acid belongs to organic acids, 3-aminosalicylic acid, hippuric acid, and N-acetyltryptophan belong to benzene ring compounds, D-glucose belongs to carbohydrates, hydroxocobalamin belongs to corrins, riboflavin-5'-phosphate belongs to flavin nucleotides, hydroxypyruvic acid and glycolic acid belong to hydroxy acids, 3-hydroxy-2-butanone belongs to organic oxygen compounds, betaine belongs to pyrimidines, and xanthurenic acid belongs to quinoline derivatives. These results suggest that there are abnormalities in multiple metabolic pathways such as amino oxides, organic acids, benzene ring compounds, carbohydrates, corrins, nucleotides, hydroxy acids, organic oxides, pyrimidines, and quinoline derivatives in patients with Alzheimer's disease, and changes in blood metabolite levels may reflect abnormalities in related metabolic pathways in the brains of patients with Alzheimer's disease, which is of great significance for early clinical diagnosis.
本发明以13种血液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。The present invention uses the mass spectrometry peak intensity values of 13 blood 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.
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。The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.
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:三甲胺-N-氧化物、莽草酸、3-氨基水杨酸、马尿酸、N-乙酰色氨酸、D-葡萄糖、羟钴胺、核黄素-5’-磷酸、羟基丙酮酸、羟基乙酸、3-羟基-2-丁酮、甜菜碱、黄尿酸中的任意一种或多种。A biomarker for Alzheimer's disease, characterized in that the biomarker includes any one or more of trimethylamine-N-oxide, shikimic acid, 3-aminosalicylic acid, hippuric acid, N-acetyltryptophan, D-glucose, hydroxocobalamin, riboflavin-5'-phosphate, hydroxypyruvic acid, glycolic acid, 3-hydroxy-2-butanone, betaine, and xanthuric acid.
- 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,所述生物标志物为三甲胺-N-氧化物、D-葡萄糖、莽草酸和羟基丙酮酸的组合。The Alzheimer's disease biomarker according to claim 1, characterized in that the biomarker is a combination of trimethylamine-N-oxide, D-glucose, shikimic acid and hydroxypyruvic acid.
- 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。A kit comprising a detection tool and instructions, wherein the instructions record the process and indicators for diagnosing a subject, characterized in that the detection tool consists of a tool for measuring the Alzheimer's disease biomarker according to claim 1 or 2.
- 根据权利要求3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:The kit according to claim 3, characterized in that the instructions record the following diagnostic process: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S1. determining the level of the Alzheimer's disease biomarker in a biological sample from a subject; andS2、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。S2. Based on the level of the Alzheimer's disease biomarker, assist in determining or diagnosing the presence of Alzheimer's disease or the risk of developing Alzheimer's disease with high specificity.
- 根据权利要求4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。The kit according to claim 4, characterized in that 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 group that does not suffer from Alzheimer's disease.
- 根据权利要求5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:三甲胺-N-氧化物的减少、莽草酸的减少、3-氨基水杨酸的减少、N-乙酰色氨酸的减少、D-葡萄糖的减少、核黄素-5’-磷酸的减少、羟基丙酮酸的减少、3-羟基-2-丁酮的减少、甜菜碱的减少、黄尿酸的减少、马尿酸的减少、羟钴胺的减少、羟基乙酸的减少。The kit according to claim 5, characterized in that in step S2, 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: a decrease in trimethylamine-N-oxide, a decrease in shikimic acid, a decrease in 3-aminosalicylic acid, a decrease in N-acetyltryptophan, a decrease in D-glucose, a decrease in riboflavin-5'-phosphate, a decrease in hydroxypyruvic acid, a decrease in 3-hydroxy-2-butanone, a decrease in betaine, a decrease in xanthuric acid, a decrease in hippuric acid, a decrease in hydroxycobalamin, and a decrease in glycolic acid.
- 根据权利要求3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。The kit according to claim 3 is characterized in that the instructions record that the mass spectrum peak height value and/or mass spectrum peak area value of the Alzheimer's disease biomarker is used as a diagnostic indicator.
- 根据权利要求3所述的试剂盒,其特征在于,所述生物样品选自血液。The kit according to claim 3, characterized in that the biological sample is selected from blood.
- 根据权利要求8所述的试剂盒,其特征在于,所述生物样品选自血清。The kit according to claim 8, characterized in that the biological sample is selected from serum.
- 权利要求1或2所述阿尔兹海默症生物标志物在制备The Alzheimer's disease biomarker according to claim 1 or 2 is prepared筛选治疗或预防阿尔兹海默症药物、Screening for drugs to treat or prevent Alzheimer's disease,辅助诊断受试者中阿尔兹海默症、Assisted diagnosis of Alzheimer's disease,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。And/or use in reagents or kits to assist in determining the risk of developing Alzheimer's disease in a subject.
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