WO2023040092A1 - 一种阿尔兹海默症生物标志物s-甲基-5'-硫代腺苷及其应用 - Google Patents

一种阿尔兹海默症生物标志物s-甲基-5'-硫代腺苷及其应用 Download PDF

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WO2023040092A1
WO2023040092A1 PCT/CN2021/138054 CN2021138054W WO2023040092A1 WO 2023040092 A1 WO2023040092 A1 WO 2023040092A1 CN 2021138054 W CN2021138054 W CN 2021138054W WO 2023040092 A1 WO2023040092 A1 WO 2023040092A1
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disease
alzheimer
sample
thioadenosine
methyl
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陈宇
叶涛
陈艺菁
樊颖颖
陈岳文
许进英
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中国科学院深圳先进技术研究院
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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
    • 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
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • 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

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  • the invention belongs to the field of biotechnology, and relates to an Alzheimer's disease biomarker S-methyl-5'-thioadenosine and its application.
  • AD Alzheimer's disease
  • the diagnosis method of AD is mainly combined diagnosis, which mainly includes: neuropsychological assessment, cognitive impairment test; brain senile plaque and Tau protein PET scan; brain magnetic resonance (MRI) and cerebrospinal fluid (CSF) markers, ⁇ - Amyloid, detection of phosphorylated tau protein, etc. (See Selkoe DJ.Alzheimer disease and aducanumab:adjusting our approach.Nat Rev Neurol.2019;15(7):365-6.), but because the early symptoms of AD are not obvious.
  • MRI brain magnetic resonance
  • CSF cerebrospinal fluid
  • early screening techniques for AD include positron emission tomography (PET) and detection of A ⁇ molecular levels in cerebrospinal fluid.
  • PET positron emission tomography
  • a ⁇ molecular levels in cerebrospinal fluid Surgical infection, and the reliability of the above diagnostic techniques for the early diagnosis of AD is not stable, so it is difficult to be used for early screening of AD.
  • AD neurodegenerative diseases
  • the host and intestinal flora produce a large number of metabolites in the process of metabolizing food substances.
  • the diversity and abundance of intestinal flora will have an important impact on the types and concentrations of small molecule metabolites in the body.
  • studying the relationship between the metabolic homeostasis of intestinal flora and the onset of AD and screening new AD biomarkers will help expand the basis for early diagnosis of AD, and can be combined with other markers to improve the diagnosis of AD.
  • the accuracy is helpful for early warning of diseases, pathological typing, and prediction and evaluation of developmental stages.
  • the present invention provides a biomarker for Alzheimer's disease, S-methyl-5'-thioadenosine, and its application.
  • the present invention is based on high-resolution non-targeted metabolomics Analytical technology for qualitative and quantitative analysis of metabolites in human feces.
  • S-methyl-5'-thioadenosine in feces is used as a marker of Alzheimer's disease.
  • the level of adenosine can assist in the early diagnosis of Alzheimer's disease, and has the characteristics of timeliness, convenience, high specificity and high sensitivity.
  • a biomarker of Alzheimer's disease said biomarker of Alzheimer's disease is S-methyl-5'-thioadenosine (S-Methyl-5'-thioadenosine).
  • S-methyl-5'-thioadenosine is C 11 H 15 N 5 O 3 S and the molecular weight is 297.33.
  • S-methyl-5'-thioadenosine (MTA) is a by-product in the biosynthesis of several important compounds, such as polyamine synthesis, in which L-methionine passes through S-adenosine in the reaction to release MTA -L-methionine (SAM) to consume, MTA is a strong inhibitor of polyamine biosynthesis and transmethylation reactions, and its concentration needs to be strictly regulated; in addition, MTA is found in the pathogen Pseudomonas aeruginosa, where MTA It is a by-product of the synthesis of autoinducer compounds PAI-1 and PAI-1-2.
  • the present invention conducts qualitative and quantitative analysis of fecal metabolites based on high-resolution non-targeted metabolomics analysis technology, and detects that the level of S-methyl-5'-thioadenosine in Alzheimer's disease feces samples is significantly lower than that in normal feces Samples, using S-methyl-5'-thioadenosine in feces as a biomarker of Alzheimer's disease, can help Alzheimer's disease by detecting the level of S-methyl-5'-thioadenosine in feces early diagnosis of the disease.
  • the present invention provides the application of the Alzheimer's disease biomarker as described in the first aspect in constructing an early diagnosis model of Alzheimer's disease and/or preparing an early diagnosis device for Alzheimer's disease.
  • the present invention provides an early diagnosis model of Alzheimer's disease
  • the input variable of the early diagnosis model of Alzheimer's disease includes the S-methyl-5'-thioadenosine described in the first aspect
  • the peak intensity value of the mass spectrum is the S-methyl-5'-thioadenosine described in the first aspect.
  • the output variables of the Alzheimer's disease early diagnosis model include differential expression multiples, and the calculation formula of the differential expression multiples is shown in equation (1):
  • the criteria for judging positive for Alzheimer's disease is that the differential expression factor is ⁇ 0.32.
  • the model uses the peak intensity of the mass spectrum of S-methyl-5'-thioadenosine as the input variable, and takes the differential expression fold as the output variable, which can quickly output results and fully characterizes S-methyl-5'-thioadenosine Samples with abnormal 5'-thioadenosine levels can assist in the early diagnosis of Alzheimer's disease.
  • the present invention provides an early diagnosis device for Alzheimer's disease, which includes the following units:
  • Sample preparation unit prepare the sample to be tested into a sample solution to be tested that can be used for separation by liquid chromatography;
  • Detection unit use a liquid chromatograph to separate the sample solution to be tested, use a mass spectrometer to perform data processing on the separated sample, and measure the peak intensity of the S-methyl-5'-thioadenosine mass spectrum described in the first aspect in the sample value;
  • Analysis unit input the detected peak intensity value of the mass spectrum of S-methyl-5'-thioadenosine into the early diagnosis model of Alzheimer's disease described in the third aspect for analysis;
  • Evaluation unit output the differential expression multiple corresponding to the sample, and judge whether it is positive for Alzheimer's disease.
  • each unit cooperates effectively, is simple and efficient, and can quickly complete sample processing, detection and obtain differential expression multiples, and at the same time, use rationally designed judgment criteria to detect positive results for Alzheimer's disease. Evaluation is of great significance for the early diagnosis of Alzheimer's disease.
  • the sample to be tested comprises a stool sample.
  • the preparation method of the sample solution to be tested includes adding the sample to be tested into an aqueous acetonitrile solution, centrifuging and collecting the supernatant to obtain the sample solution to be tested.
  • the preparation method of the sample solution to be tested comprises the following steps:
  • the volume ratio of methanol, acetonitrile and water in the methanol/acetonitrile/water solution is (1-2):(1-2):1 including but not limited to 1.2:2:1, 1.2:1:1, 2 :2:1, 1.4:1.5:1, 1.6:1.2:1, 1.8:2:1, 1.9:1.8:1 or 1.1:1.4:1.
  • the volume ratio of acetonitrile and water in the aqueous acetonitrile solution is (1-2):1, including but not limited to 1.1:1, 1.2:1, 1.3:1, 1.5:1, 1.6:1, 1.7:1 , 1.8:1 or 1.9:1.
  • the liquid chromatograph includes an ultra-high performance liquid chromatograph.
  • the ultra-high performance liquid chromatograph comprises Agilent 1290 Infinity LC ultra-high performance liquid chromatograph.
  • the mass spectrometer comprises a tandem time-of-flight mass spectrometer.
  • the tandem time-of-flight mass spectrometer includes AB Triple TOF 6600 mass spectrometer.
  • the data processing includes:
  • tandem time-of-flight mass spectrometer uses the tandem time-of-flight mass spectrometer to collect the first-order spectrum and second-order spectrum of the separated sample, and convert the first-order spectrum and second-order spectrum into mzXML format, and then perform peak alignment and retention time For calibration, peak area extraction and structure identification, determine the peak intensity value of the mass spectrum of S-methyl-5'-thioadenosine described in the first aspect in the sample.
  • the Alzheimer's disease early diagnosis device includes the following units:
  • Sample preparation unit prepare the sample to be tested into a sample solution to be tested that can be used for separation by liquid chromatography;
  • Detection unit use the liquid chromatograph to separate the sample solution to be tested, use a tandem time-of-flight mass spectrometer to collect the first-order spectrum and second-order spectrum of the separated sample, and collect the first-order spectrum
  • the figure and the secondary spectrogram are converted into mzXML format, and then peak alignment, retention time correction, peak area extraction and structural identification are performed, and the S-methyl-5'-thioadenosine mass spectrum described in the first aspect is determined in the sample. peak intensity value;
  • Analysis unit input the detected peak intensity value of the mass spectrum of S-methyl-5'-thioadenosine into the early diagnosis model of Alzheimer's disease described in the third aspect for analysis;
  • Evaluation unit output the differential expression multiple corresponding to the sample, and judge whether it is positive for Alzheimer's disease.
  • the detection of the level of S-methyl-5'-thioadenosine in the stool sample can be used as a diagnostic basis, combined with other detection results, to assist in the early diagnosis of Alzheimer's disease, and it is expected to improve Alzheimer's disease.
  • the accuracy of the diagnosis of Alzheimer's disease but it cannot be used alone as a diagnostic indicator for 100% diagnosis of Alzheimer's disease.
  • S-Methyl-5'-thioadenosine belongs to the cysteine and methionine metabolism (Cysteine and methionine metabolism) pathway in the amino acid metabolism pathway (Amino acid metabolism pathway).
  • the present invention provides the application of the Alzheimer's disease biomarker described in the first aspect in screening drugs for treating and/or preventing Alzheimer's disease.
  • the Alzheimer's disease biomarker described in the first aspect is used as a target to screen a drug for treating and/or preventing Alzheimer's disease.
  • the present invention has the following beneficial effects:
  • the present invention detects for the first time that the level of S-methyl-5'-thioadenosine in the stool sample of Alzheimer's disease is significantly lower than that of normal stool samples, and the S-methyl-5'-thioadenosine in the stool is used as Al
  • the biomarker of Alzheimer's disease can assist the early diagnosis of Alzheimer's disease by detecting the level of S-methyl-5'-thioadenosine in feces, which is helpful for non-invasive and rapid detection, and is timely, convenient and highly specific properties of high sensitivity and high sensitivity.
  • Fig. 1 is the level figure of S-methyl-5'-thioadenosine in the stool sample of AD model mouse and wild-type mouse;
  • Figure 2 is a level map of 1-Aminocyclopropanecarboxylic acid in the cerebral cortex samples of AD model mice and wild-type mice;
  • Figure 3 is a graph showing the levels of 3-Phospho-D-glycerate in the cerebral cortex samples of AD model mice and wild-type mice.
  • the feces of 10 AD model mice and 10 wild-type mice cultured under the same conditions were collected respectively, and the samples of wild-type mice were numbered FWT-1-1 ⁇ FWT-1-10 in turn, and the samples of AD model mice were The samples are numbered FTG-1-1 ⁇ FTG-1-10 in turn, and the level of S-methyl-5'-thioadenosine in the samples is detected by ultra-high performance liquid chromatography-tandem time-of-flight mass spectrometry.
  • the specific methods include :
  • AB Triple TOF 6600 mass spectrometer is used to collect the first-order and second-order spectra of the samples separated by ultra-high performance liquid chromatography in step (2).
  • the ESI source conditions are as follows: Ion Source Gas1 (Gas1): 60 , Ion Source Gas2 (Gas2): 60, Curtain gas (CUR): 30, source temperature: 600°C, IonSapary Voltage Floating (ISVF) ⁇ 5500V (both positive and negative modes); TOF MS scan m/z range: 60 ⁇ 1000Da, product ion scan m/z range: 25 ⁇ 1000Da, TOF MS scan accumulation time 0.20s/spectra, product ion scan accumulation time 0.05s/spectra; the secondary mass spectrum is obtained by information dependent acquisition (IDA) and high sensitivity Mode, Declustering potential (DP): ⁇ 60V (both positive and negative modes), Collision Energy: 35 ⁇ 15eV, IDA settings are as follows: Exclude isotopes within 4Da, Candidat
  • S-methyl-5'-thioadenosine belongs to the Cysteine and methionine metabolism (cysteine and methionine metabolism) pathway in the amino acid metabolism pathway (Amino acid metabolism pathway)
  • Cysteine is an aliphatic thiol-containing polar ⁇ -amino acid. It is a conditionally essential amino acid and a sugar-forming amino acid for the human body. It can be converted from methionine in the body and can be converted with cystine.
  • cysteine Under anaerobic conditions, it is decomposed into pyruvate, hydrogen sulfide and ammonia through the action of desulfurase, or through transamination, it is decomposed into pyruvate and sulfur through the intermediate product ⁇ -mercaptopyruvate, and under oxidative conditions, it is oxidized into Cysteine sulfite can be decomposed into pyruvic acid and sulfurous acid through transamination, and can be decomposed into hypotaurine and taurine through decarboxylation; methionine, also known as methionine, is a human body’s One of the essential amino acids, the lack of which will lead to the blockage of protein synthesis in the body, methionine can promote the methylation of phospholipids in the liver cell membrane, and enhance the membrane fluidity.
  • the Na + , K + -ATPase pump has a strong effect and can reduce the bile in the liver cells.
  • the stasis of the liver, the transsulfurization effect is strengthened, thereby enhancing the synthesis of cysteine, glutathione and taurine in the liver cells, reducing the accumulation of bile acids in the liver, and strengthening the detoxification effect.
  • the cerebral cortex samples of 10 AD model mice and 10 wild-type mice cultured under the same conditions were collected respectively.
  • the mouse cerebral cortex samples were sequentially numbered CTG-1-1 ⁇ CTG-1-10, and the metabolites were qualitatively and quantitatively analyzed by ultra-high performance liquid chromatography-tandem time-of-flight mass spectrometry, and the specific methods included:
  • the VIP value and p-value are comprehensively considered to screen the significant differential metabolites.
  • the results are shown in Figure 2, Figure 3 and Table 2, AD
  • the level of 1-aminocyclopropanecarboxylic acid (1-Aminocyclopropanecarboxylic acid) in the cerebral cortex of model mice was significantly higher than that of wild-type mice, while the level of 3-phosphoglycerate (3-Phospho-D-glycerate) was significantly lower than that of wild-type mice mice, and the above-mentioned metabolites all belong to the Cysteine and methionine metabolism pathway in the Amino acid metabolism pathway, indicating that the Cysteine and methionine metabolism pathway in the Amino acid metabolism pathway is related to Alzheimer's disease, and the present invention finds that S-methionine metabolism
  • the base-5'-thioadenosine also belongs to the Cysteine and methionine metabolism pathway in the Amino acid metabolism pathway, which indicates from another aspect that the changes in the level of S-methyl-5
  • the present invention detects for the first time that the level of S-methyl-5'-thioadenosine in the stool samples of Alzheimer's disease is significantly lower than that in normal stool samples, and the level of S-methyl-5'-thioadenosine in the stool
  • adenosine can assist in the early diagnosis of Alzheimer's disease by detecting the level of S-methyl-5'-thioadenosine in feces. , convenience, high specificity and high sensitivity.
  • the present invention illustrates the detailed methods of the present invention through the above-mentioned examples, but the present invention is not limited to the above-mentioned detailed methods, that is, it does not mean that the present invention must rely on the above-mentioned detailed methods to be implemented.
  • Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

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Abstract

一种阿尔兹海默症生物标志物S-甲基-5'-硫代腺苷及其应用。阿尔兹海默症生物标志物为S-甲基-5'-硫代腺苷。首次检测到阿尔兹海默症粪便样本中S-甲基-5'-硫代腺苷水平显著低于正常粪便样本，将粪便中S-甲基-5'-硫代腺苷作为阿尔兹海默症生物标志物，通过检测粪便中S-甲基-5'-硫代腺苷水平能够辅助阿尔兹海默症早期诊断，有助于无创快速检测，且具备及时、方便、高特异性及高灵敏度的特点。

Description

一种阿尔兹海默症生物标志物S-甲基-5′-硫代腺苷及其应用 技术领域
本发明属于生物技术领域，涉及一种阿尔兹海默症生物标志物S-甲基-5'-硫代腺苷及其应用。
背景技术
阿尔兹海默症(Alzheimer disease，AD)是一种起病隐匿的进行性发展的神经系统退行性疾病，临床上以记忆障碍、失语、失用、失认、视空间技能损害、执行功能障碍以及人格和行为改变等表现为特征。
目前AD的诊断方法主要是联合诊断，主要包括：神经心理学评估，认知损伤测试；脑部老年斑块和Tau蛋白PET扫描；脑部核磁共振(MRI)和脑脊液(CSF)标志物，β-淀粉样蛋白，磷酸化tau蛋白检测等(参见Selkoe DJ.Alzheimer disease and aducanumab:adjusting our approach.Nat Rev Neurol.2019；15(7):365-6.)，但由于AD早期症状不明显，当对病症做出明确诊断时，患者多到达病程晚期，多数神经元出现死亡，如果能在病人发病早期进行快速诊断或使潜在病人提前发现风险，针对性地进行治疗或预防，能有助于患者病程滞留在轻微智力损伤阶段(mild cognitive impairment，MCI)而减缓恶化，从而保证病人生活质量和减轻社会负担。
现阶段对AD的早期筛查技术包括正电子发射型计算机断层显像(PET)和脑脊液Aβ分子水平检测等，前者要对受检者注射一定剂量的放射性物质，后者操作损伤大，易造成外科感染，且上述诊断技术针对AD早期诊断的可靠性也不稳定，因此很难用于AD早期筛查。
因此，对AD早期诊断新的标志物开发是未来对AD诊疗的重要方向之一。
有研究发现多种神经精神疾病如帕金森、抑郁症、自闭症等与肠道菌群失衡有关，并且80％以上AD患者存在肠道菌群失衡的现象，提示肠道菌群稳态与AD等神经退行性疾病的发病进程密切相关。宿主和肠道菌群在代谢食物物质的过程中产生大量的代谢物，肠道菌群的多样性以及丰度的改变会对机体中小分子代谢物的种类和浓度产生重要影响，有数据显示AD患者肠道菌群组成与健康同龄人不同，且越来越多证据表明各种代谢途径的紊乱可能介导AD的病理发生和发展，AD中的菌群失衡可能与机体代谢紊乱的发生有重要关联，而机体外周代谢改变又可能通过血液循环进一步导致中枢神经系统代谢紊乱。
综上所述，研究肠道菌群代谢稳态与AD发病的关系，筛选新的AD生物标志物，有助于扩充AD早期诊断的判断依据，可与其他标志物检测相互结合，提高AD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。
发明内容
针对现有技术的不足和实际需求，本发明提供一种阿尔兹海默症生物标志物S-甲基-5'-硫代腺苷及其应用，本发明基于高分辨非靶向代谢组学分析技术对人粪便代谢物进行定性定量分析，首次将粪便中S-甲基-5'-硫代腺苷作为阿尔兹海默症标志物，通过检测粪便中S-甲基-5'-硫代腺苷水平能够辅助阿尔兹海默症早期诊断，且具备及时、方便、高特异性及高灵敏度的特点。
为达上述目的，本发明采用以下技术方案：
第一方面，一种阿尔兹海默症生物标志物，所述阿尔兹海默症生物标志物为S-甲基-5'-硫代腺苷(S-Methyl-5'-thioadenosine)。
S-甲基-5'-硫代腺苷化学式为C 11H 15N 5O 3S，分子量为297.33。S-甲基-5'-硫 代腺苷(MTA)是几种重要化合物生物合成中的副产物，如多胺合成，其中L-甲硫氨酸在释放MTA的反应中通过S-腺苷-L-甲硫氨酸(SAM)来消耗，MTA是多胺生物合成和转甲基反应的强抑制剂，其浓度需要严格调节；此外，在病原体铜绿假单胞菌中发现MTA，其中MTA是合成自诱导剂化合物PAI-1和PAI-1-2的副产物。
本发明基于高分辨非靶向代谢组学分析技术对粪便代谢物进行定性定量分析，检测到阿尔兹海默症粪便样本中S-甲基-5'-硫代腺苷水平显著低于正常粪便样本，将粪便中S-甲基-5'-硫代腺苷作为阿尔兹海默症生物标志物，通过检测粪便中S-甲基-5'-硫代腺苷水平能够辅助阿尔兹海默症早期诊断。
第二方面，本发明提供如第一方面所述的阿尔兹海默症生物标志物在构建阿尔兹海默症早期诊断模型和/或制备阿尔兹海默症早期诊断装置中的应用。
第三方面，本发明提供一种阿尔兹海默症早期诊断模型，所述阿尔兹海默症早期诊断模型的输入变量包括第一方面所述的S-甲基-5'-硫代腺苷质谱的峰强度值。
优选地，所述阿尔兹海默症早期诊断模型的输出变量包括差异表达倍数，所述差异表达倍数的计算公式如方程式(1)所示：
Figure PCTCN2021138054-appb-000001
优选地，阿尔兹海默症阳性的判断标准为所述差异表达倍数≤0.32。
本发明中，通过对正常粪便样本和AD粪便样本中S-甲基-5'-硫代腺苷质谱的峰强度值进行充分对比分析，并进行理性设计，构建了一种阿尔兹海默症早期诊断模型，所述模型以S-甲基-5'-硫代腺苷质谱的峰强度值为输入变量，以差异表达倍数为输出变量，能够快速输出结果，且充分表征S-甲基-5'-硫代腺苷水 平异常的样本，从而辅助阿尔兹海默症早期诊断。
第四方面，本发明提供一种阿尔兹海默症早期诊断装置，所述装置包括如下单元：
样本配制单元：将待测样本配制成可用于液相色谱仪分离的待测样本溶液；
检测单元：利用液相色谱仪分离待测样本溶液，利用质谱仪对分离后样本进行数据处理，测定样本中第一方面所述的S-甲基-5'-硫代腺苷质谱的峰强度值；
分析单元：将检测到的S-甲基-5'-硫代腺苷质谱的峰强度值输入第三方面所述的阿尔兹海默症早期诊断模型进行分析；
评估单元：输出样本对应的差异表达倍数，并判断是否为阿尔兹海默症阳性。
本发明的阿尔兹海默症早期诊断装置中，各单元间有效配合，简单高效，能够快速完成样本处理、检测及获得差异表达倍数，同时以经过合理设计的判断标准进行阿尔兹海默症阳性评估，对于阿尔兹海默症早期诊断具有重要意义。
优选地，所述待测样本包括粪便样本。
优选地，所述待测样本溶液的配制方法包括将待测样本加入乙腈水溶液中，离心并收集上清液，得到所述待测样本溶液。
优选地，所述待测样本溶液的配制方法包括以下步骤：
(1)取待测样本加入预冷甲醇/乙腈/水溶液中，混合并超声25～35min(例如可以是26min、27min、28min、29min或32min)，置于-20～-15℃(例如可以是-19℃、-18℃、-16℃或-17℃)静置5～15min(例如可以是6min、7min、8min、9min、10min、12min或14min)，于0～4℃(例如可以是1℃、2℃或3℃)、12000～16000×g(例如可以是12200×g、12400×g、12600×g、12800×g、 13200×g、12600×g、15000×g或15800×g)离心15～25min(例如可以是16min、17min、18min、19min、20min、21min、22min、23min或24min)，取上清进行真空干燥，得到预处理样本；
(2)将所述预处理样本加入80～120μL乙腈水溶液中复溶，涡旋，于0～4℃、12000～16000×g(例如可以是12200×g、12400×g、12600×g、12800×g、13200×g、12600×g、15000×g或15800×g)离心10～20min(例如可以是11min、12min、13min、14min、15min、16min、17min、18min或19min)，取上清液，得到所述待测样本溶液。
优选地，所述甲醇/乙腈/水溶液中甲醇、乙腈和水的体积比为(1～2):(1～2):1包括但不限于1.2:2:1、1.2:1:1、2:2:1、1.4:1.5:1、1.6:1.2:1、1.8:2:1、1.9:1.8:1或1.1:1.4:1。
优选地，所述乙腈水溶液中乙腈和水的体积比为(1～2):1，包括但不限于1.1:1、1.2:1、1.3:1、1.5:1、1.6:1、1.7:1、1.8:1或1.9:1。
优选地，所述液相色谱仪包括超高效液相色谱仪。
优选地，所述超高效液相色谱仪包括Agilent 1290 Infinity LC超高效液相色谱仪。
优选地，所述质谱仪包括串联飞行时间质谱联用仪。
优选地，所述串联飞行时间质谱联用仪包括AB Triple TOF 6600质谱仪。
优选地，所述数据处理包括：
利用串联飞行时间质谱联用仪对分离后样本进行一级谱图和二级谱图的采集，并将所述一级谱图和二级谱图转换成mzXML格式，然后进行峰对齐、保留时间校正、提取峰面积以及结构鉴定，测定样本中第一方面所述的S-甲基-5'- 硫代腺苷质谱的峰强度值。
作为优选的技术方案，所述阿尔兹海默症早期诊断装置包括如下单元：
样本配制单元：将待测样本配制成可用于液相色谱仪分离的待测样本溶液；
检测单元：利用所述液相色谱仪分离所述待测样本溶液，利用串联飞行时间质谱联用仪对分离后样本进行一级谱图和二级谱图的采集，并将所述一级谱图和二级谱图转换成mzXML格式，然后进行峰对齐、保留时间校正、提取峰面积以及结构鉴定，测定样本中第一方面所述的S-甲基-5'-硫代腺苷质谱的峰强度值；
分析单元：将检测到的S-甲基-5'-硫代腺苷质谱的峰强度值输入第三方面所述的阿尔兹海默症早期诊断模型进行分析；
评估单元：输出样本对应的差异表达倍数，并判断是否为阿尔兹海默症阳性。
本发明中，对粪便样本中S-甲基-5'-硫代腺苷水平进行检测，可以作为一种诊断依据，与其他检测结果结合，辅助阿尔兹海默症早期诊断，预期可以提高阿尔兹海默症诊断的准确性，但并不能单独作为能够100％诊断阿尔兹海默症的诊断指标。
本发明中，通过KEGG通路分析发现S-Methyl-5'-thioadenosine属于氨基酸合成代谢通路(Amino acid metabolism pathway)中的半胱氨酸和蛋氨酸代谢(Cysteine and methionine metabolism)途径。
第五方面，本发明提供第一方面所述的阿尔兹海默症生物标志物在筛选治疗和/或预防阿尔兹海默症的药物中的应用。
即以第一方面所述的阿尔兹海默症生物标志物作为靶点筛选治疗和/或预 防阿尔兹海默症的药物。
与现有技术相比，本发明具有以下有益效果：
本发明首次检测到阿尔兹海默症粪便样本中S-甲基-5'-硫代腺苷水平显著低于正常粪便样本，将粪便中S-甲基-5'-硫代腺苷作为阿尔兹海默症生物标志物，通过检测粪便中S-甲基-5'-硫代腺苷水平能够辅助阿尔兹海默症早期诊断，有助于无创快速检测，且具备及时、方便、高特异性及高灵敏度的特点。
附图说明
图1为AD模型小鼠和野生型小鼠的粪便样本中S-甲基-5'-硫代腺苷水平图；
图2为AD模型小鼠和野生型小鼠的大脑皮层样本中1-Aminocyclopropanecarboxylic acid水平图；
图3为AD模型小鼠和野生型小鼠的大脑皮层样本中3-Phospho-D-glycerate水平图。
具体实施方式
为进一步阐述本发明所采取的技术手段及其效果，以下结合实施例和附图对本发明作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本发明，而非对本发明的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1
本实施例对9月龄AD模型小鼠(APP/PS1转基因小鼠，由南京大学模式动物研究所提供)与野生型(WT)小鼠的粪便样本进行代谢物定性定量分析。
分别采集在相同条件下培养的10只AD模型小鼠和10只野生型小鼠的粪便，将野生型小鼠样本依次编号为FWT-1-1～FWT-1-10，将AD模型小鼠样本依次编号为FTG-1-1～FTG-1-10，采用超高效液相色谱-串联飞行时间质谱联用检测样本中的S-甲基-5'-硫代腺苷水平，具体方法包括：
(1)取粪便样本加入预冷甲醇/乙腈/水溶液(体积比为2:2:1)，涡旋混合，低温超声30min，置于-20℃静置10min，于4℃、14000×g离心20min，取上清进行真空干燥，得到预处理样本；
(2)将所述预处理样本加入100μL乙腈水溶液(体积比为乙腈:水＝1:1)中复溶，涡旋，于4℃、14000×g离心15min，取上清液进样分析，采用Agilent 1290 Infinity LC超高效液相色谱系统(UHPLC)HILIC色谱柱进行分离，柱温25℃；流速0.5mL/min；进样量2μL；流动相组成包括：A相：乙酸铵和氨水混合的水溶液(乙酸铵和氨水终浓度均为25mM)，B相：乙腈；梯度洗脱程序如下：0～0.5min，95％B相；0.5～7min，B相从95％线性变化至65％；7～8min，B相从65％线性变化至40％；8～9min，B相维持在40％；9～9.1min，B相从40％线性变化至95％；9.1～12min，B相维持在95％；整个分析过程中样本置于4℃自动进样器中；
(3)采用AB Triple TOF 6600质谱仪对步骤(2)超高效液相色谱系分离后的样本进行样本一级、二级谱图的采集，ESI源条件如下：Ion Source Gas1(Gas1)：60，Ion Source Gas2(Gas2)：60，Curtain gas(CUR)：30，source temperature：600℃，IonSapary Voltage Floating(ISVF)±5500V(正负两种模式)；TOF MS scan m/z range：60～1000Da，product ion scan m/z range：25～1000Da，TOF MS scan accumulation time 0.20s/spectra，product ion scan accumulation  time 0.05s/spectra；二级质谱采用information dependent acquisition(IDA)获得，并且采用high sensitivity模式，Declustering potential(DP)：±60V(正负两种模式)，Collision Energy：35±15eV，IDA设置如下Exclude isotopes within 4Da，Candidate ions to monitor per cycle：10，将采集到的Wiff格式的原始数据经ProteoWizard转换成mzXML格式，然后采用XCMS软件进行峰对齐、保留时间校正和提取峰面积，对XCMS提取得到的数据进行代谢物结构鉴定，并分析样本中的S-甲基-5'-硫代腺苷水平，利用R语言工具(R package(ropls))进行变异倍数分析(Fold Change Analysis，FC Analysis)、主成分分析(PCA)、正交偏最小二乘法判别分析(OPLS-DA)和T检验(Student's t-test)，结果如图1及表1所示，AD模型小鼠粪便样本中S-甲基-5'-硫代腺苷水平显著低于野生型小鼠，表明可将粪便中S-甲基-5'-硫代腺苷作为阿尔兹海默症生物标志物，通过检测粪便中S-甲基-5'-硫代腺苷水平能够辅助阿尔兹海默症早期诊断。
表1
Figure PCTCN2021138054-appb-000002
注：*为p<0.05。
此外，进一步通过KEGG通路注释与分析发现S-甲基-5'-硫代腺苷属于氨基酸合成代谢通路(Amino acid metabolism pathway)中的Cysteine and methionine metabolism(半胱氨酸和蛋氨酸代谢)途径，半胱氨酸是脂肪族含巯基的极性α-氨基酸，是人体的条件必需氨基酸和生糖氨基酸，可由体内的蛋氨酸转化而来，可与胱氨酸互相转化，半胱氨酸的分解是在厌氧条件下通过脱硫 氢酶的作用分解成丙酮酸和硫化氢和氨，或是通过转氨基作用，经由中间产物β-巯基丙酮酸分解成为丙酮酸和硫黄，在氧化条件下，氧化成半胱氨酸亚硫酸后，可经转氨基作用分解成为丙酮酸与亚硫酸，以及由脱羧基作用分解成为亚牛磺酸、牛磺酸等；蛋氨酸，也称甲硫氨酸，是人体的必需氨基酸之一，其缺乏会导致体内蛋白质合成受阻，甲硫氨酸可以促进肝细胞膜磷脂甲基化，使膜流动性增强Na +、K +-ATP酶泵作用强，可以减少肝细胞内胆汁的淤积，转硫基作用加强，从而增强了肝细胞内半胱氨酸、谷胱苷肽及牛磺酸的合成，减少了胆汁酸在肝内聚积，加强了解毒作用。
实施例2
本实施例对9月龄AD模型小鼠与野生型(WT)小鼠的大脑皮层样本进行代谢物定性定量分析。
分别采集在相同条件下培养的10只AD模型小鼠和10只野生型小鼠的大脑皮层样本，将野生型大脑皮层样本依次编号为CWT-1-1～CWT-1-10，将AD模型小鼠大脑皮层样本依次编号为CTG-1-1～CTG-1-10，采用超高效液相色谱-串联飞行时间质谱联用仪进行代谢物定性定量分析，具体方法包括：
(1)将小鼠断颈处死后用手术剪剖开脑壳，暴露大脑，将大脑沿中间脑缝剖成两半，分别去掉小脑、脑干、丘脑、下皮层和海马体，剩下大脑皮层，收集左右两边完整的大脑皮层作为样本，加入预冷甲醇/乙腈/水溶液(体积比为2:2:1)，涡旋混合，低温超声30min，置于-20℃静置10min，于4℃、14000×g离心20min，取上清进行真空干燥，得到预处理样本；
(2)将所述预处理样本加入100μL乙腈水溶液(体积比为乙腈:水＝1:1)中复溶，涡旋，于4℃、14000×g离心15min，取上清液进样分析，采用Agilent  1290 Infinity LC超高效液相色谱系统(UHPLC)HILIC色谱柱进行分离，条件与实施例1相同；
(3)采用AB Triple TOF 6600质谱仪对步骤(2)超高效液相色谱系分离后的样本进行样本一级、二级谱图的采集，条件与实施例1相同，将采集到的Wiff格式的原始数据经ProteoWizard转换成mzXML格式，然后采用XCMS软件进行峰对齐、保留时间校正和提取峰面积，对XCMS提取得到的数据进行代谢物结构鉴定，随后进行单变量统计分析、多维统计分析、差异代谢物筛选、差异代谢物相关性分析和KEGG通路分析，其中，OPLS-DA模型得到的变量权重值(Variable Importance for the Projection，VIP)能够用于衡量各代谢物的表达模式对各组样本分类判别的影响强度和解释能力，挖掘具有生物学意义的差异分子，本实施例综合考虑VIP值和p-value来筛选显著性差异代谢物，结果如图2、图3以及表2所示，AD模型小鼠大脑皮层组织的1-氨基环丙羧酸(1-Aminocyclopropanecarboxylic acid)水平显著高于野生型小鼠，而3-磷酸甘油酸(3-Phospho-D-glycerate)水平显著低于野生型小鼠，且上述代谢物均属于Amino acid metabolism pathway中的Cysteine and methionine metabolism途径，说明Amino acid metabolism pathway中的Cysteine and methionine metabolism途径与阿尔兹海默症存在关联性，而本发明发现S-甲基-5'-硫代腺苷也属于Amino acid metabolism pathway中的Cysteine and methionine metabolism途径，从另一层面表明粪便代谢物中S-甲基-5'-硫代腺苷水平变化可能反映AD脑中相关代谢通路的异常，对临床早期诊断具有重要意义。
表2
Figure PCTCN2021138054-appb-000003
Figure PCTCN2021138054-appb-000004
注：*为p<0.05，**为p<0.01。
综上所述，本发明首次检测到阿尔兹海默症粪便样本中S-甲基-5'-硫代腺苷水平显著低于正常粪便样本，将粪便中S-甲基-5'-硫代腺苷作为阿尔兹海默症生物标志物，通过检测粪便中S-甲基-5'-硫代腺苷水平能够辅助阿尔兹海默症早期诊断，有助于无创快速检测，且具备及时、方便、高特异性及高灵敏度的特点。
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (10)

  1. 一种阿尔兹海默症生物标志物，其特征在于，所述阿尔兹海默症生物标志物为S-甲基-5'-硫代腺苷。
  2. 如权利要求1所述的阿尔兹海默症生物标志物在构建阿尔兹海默症早期诊断模型和/或制备阿尔兹海默症早期诊断装置中的应用。
  3. 一种阿尔兹海默症早期诊断模型，其特征在于，所述阿尔兹海默症早期诊断模型的输入变量包括权利要求1所述的S-甲基-5'-硫代腺苷质谱的峰强度值。
  4. 根据权利要求3所述的阿尔兹海默症早期诊断模型，其特征在于，所述阿尔兹海默症早期诊断模型的输出变量包括差异表达倍数，所述差异表达倍数的计算公式如方程式(1)所示：
    Figure PCTCN2021138054-appb-100001
  5. 根据权利要求4所述的阿尔兹海默症早期诊断模型，其特征在于，阿尔兹海默症阳性的判断标准为所述差异表达倍数≤0.32。
  6. 一种阿尔兹海默症早期诊断装置，其特征在于，所述装置包括如下单元：
    样本配制单元：将待测样本配制成可用于液相色谱仪分离的待测样本溶液；
    检测单元：利用所述液相色谱仪分离所述待测样本溶液，利用质谱仪对分离后样本进行数据处理，测定样本中权利要求1所述的S-甲基-5'-硫代腺苷质谱的峰强度值；
    分析单元：将检测到的S-甲基-5'-硫代腺苷质谱的峰强度值输入权利要求3-5任一项所述的阿尔兹海默症早期诊断模型进行分析；
    评估单元：输出样本对应的差异表达倍数，并判断是否为阿尔兹海默症阳性。
  7. 根据权利要求6所述的装置，其特征在于，所述待测样本包括粪便样本。
  8. 根据权利要求6或7所述的装置，其特征在于，所述数据处理包括：
    利用串联飞行时间质谱联用仪对分离后样本进行一级谱图和二级谱图的采集，并将所述一级谱图和二级谱图转换成mzXML格式，然后进行峰对齐、保留时间校正、提取峰面积以及结构鉴定，测定样本中权利要求1所述的S-甲基-5'-硫代腺苷质谱的峰强度值。
  9. 根据权利要求6-8任一项所述的装置，其特征在于，所述装置包括如下单元：
    样本配制单元：将待测样本配制成可用于液相色谱仪分离的待测样本溶液；
    检测单元：利用所述液相色谱仪分离所述待测样本溶液，利用串联飞行时间质谱联用仪对分离后样本进行一级谱图和二级谱图的采集，并将所述一级谱图和二级谱图转换成mzXML格式，然后进行峰对齐、保留时间校正、提取峰面积以及结构鉴定，测定样本中权利要求1所述的S-甲基-5'-硫代腺苷质谱的峰强度值；
    分析单元：将检测到的S-甲基-5'-硫代腺苷质谱的峰强度值输入权利要求3-5任一项所述的阿尔兹海默症早期诊断模型进行分析；
    评估单元：输出样本对应的差异表达倍数，并判断是否为阿尔兹海默症阳性。
  10. 权利要求1所述的阿尔兹海默症生物标志物在筛选治疗和/或预防阿尔兹海默症的药物中的应用。
PCT/CN2021/138054 2021-09-17 2021-12-14 一种阿尔兹海默症生物标志物s-甲基-5'-硫代腺苷及其应用 WO2023040092A1 (zh)

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CN102573854A (zh) * 2009-06-11 2012-07-11 普罗耶克托生物医学Cima有限公司 5’-甲硫腺苷的神经保护特性
CN109709235A (zh) * 2019-02-25 2019-05-03 马红华 阿尔茨海默病或轻度老年认知障碍的早期诊断、预测生物标志物组合、应用及其测定方法
CN110944650A (zh) * 2017-03-31 2020-03-31 轴向生物治疗股份有限公司 用于治疗和预防孤独症及相关障碍的肠选择性螯合剂
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CN102573854A (zh) * 2009-06-11 2012-07-11 普罗耶克托生物医学Cima有限公司 5’-甲硫腺苷的神经保护特性
CN110944650A (zh) * 2017-03-31 2020-03-31 轴向生物治疗股份有限公司 用于治疗和预防孤独症及相关障碍的肠选择性螯合剂
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