Abstract
Objective
To investigate the metabolic profiles of urine from
APP/PS1
mice with early-stage Alzheimer's disease (AD).
Methods
Urine samples were collected from 13
APP/PS1
mice of 16 weeks and 15 wild-type mice.
1
H-NMR spectroscopy was acquired with a one-dimensional NOESY pulse sequence, and the integral values were imported to SIMCA-P+12.0 software for analysis.
Results
The metabonomic analysis showed that the metabolic profiles of the
APP/PS1
mice were significantly different from that of age-matched wild-type mice. The levels of 3-hydroxybutyrate, 2-hydroxybutyrate, succinic acid, 2-ketoglutaric acid, citric acid, cis-aconitic acid, fumaric acid decreased, and those of acetic acid, trimethylamine, taurine, creatinine, hippuric acid, formic acid, trigonelline, urea increased (all
P
< 0.05).
Conclusion
Metabolic pathways including glucose metabolism and methylamine metabolism may be involved in the pathogenesis of early AD.
Keywords:
Alzheimer disease/diagnosis, Alzheimer Disease/physiopathology, Urine, Energy metabolism, Methylamines/metabolism, Magnetic resonance spectroscopy, Early diagnosis, Disease models, animal
阿尔茨海默病(Alzheimer's disease，AD)是一种严重的神经退行性疾病，其主要病理特征为老年斑和神经原纤维缠结
。据WHO报道，目前全球共有痴呆症患者约3560万，以AD患者为主
。在我国60岁以上人群中，AD的患病率为1.6%
。目前临床上AD的诊断主要根据患者的病史、体征、症状、实验室检查和辅助检查进行综合分析，而确诊需要脑神经病理检测，无较好的早期诊断方法
疾病的病理变化往往会导致机体的基础代谢产生相应改变，从而引起小分子代谢物浓度或种类发生变化，最终造成与健康个体之间代谢谱的差异，而这种差异往往早于临床症状出现。因此，利用代谢组学技术研究患者与健康个体间代谢谱的差异，找到与疾病密切相关的生物学标志物，是实现疾病早期诊断的可行方法
。核磁共振波谱法是一种常见的代谢组学分析方法，因其样品制备简单、检测快速、无破坏性，且具有高重现性，所以广泛应用于疾病的早期诊断
APP/PS1
双转基因小鼠含有人
APP
swedish突变位点和人
PS1
ΔE9突变位点，该模型3月龄出现学习记忆缺陷，5月龄开始出现老年斑，具有与AD相似的病理表型，因此被广泛应用于AD研究
。本研究运用核磁共振波谱检测结合多变量模式识别技术对
APP/PS1
小鼠的尿液进行代谢组学研究，分析潜在的代谢标志物，为阐明AD的发病机制提供重要线索，并为临床早期诊治提供新的思路。
APP/PS1
双转基因小鼠(
n
=13)以及野生型小鼠(
n
=15)购于南京大学模式动物研究所，饲养于温州医科大学动物实验中心SPF级动物饲养房。动物饲养房从早上8 :00开始12 h交替照明，温度为25~27 ℃，自由摄食饮水。所有操作程序严格遵守美国国立卫生研究院的实验动物饲养和使用手册
三甲硅烷基丙磺酸钠(TSP)为美国Sigma公司产品；重水(99.9%氘代)购于美国剑桥同位素实验室。纯水由Milli-Q超纯水系统(美国Millipore公司)生产。低温离心机5415R为德国Eppendorf公司产品；漩涡仪为美国Thermo公司产品；血糖试纸及血糖仪购自贝朗医疗(上海)国际贸易有限公司；Bruker AVANCE Ⅲ 600 MHz超导高分辨核磁共振波谱仪为德国Bruker公司产品。
16周周龄小鼠禁食12 h后用代谢笼收集12 h的尿液(8:00至20 :00)，尿液置于冰中保持低温，1000×
g
，4 ℃低温离心5 min，取上清液，保存于-80 ℃冰箱。
将尿液样本解冻，随后在150 μL尿液中加入350 μL磷酸钠缓冲液(0.2 mol/L，酸碱度值为7.4)以减少因酸碱度值变化引起的化学位移差异，并加入50 μL含TSP的重水(质量浓度为0.36 mg/mL)锁场，TSP用于定标。在4 ℃条件下12 000×
g
离心10 min去除样本中的沉淀后，取500 μL上清液移入直径为5 mm的样品管中。尿液核磁共振氢谱的采集在Bruker AVANCE Ⅲ 600 MHz超导高分辨核磁共振波谱仪中完成，
1
H的共振频率为600.13 MHz，配有三共振探头和Z轴的脉冲场梯度，采样温度为24.85 ℃(298.0 K)。利用一维NOESY脉冲序列采集尿液样本的核磁共振氢谱，谱宽12 000 Hz，采样点数为32 K，弛豫时间为4 s，采样时间为2.66 s；在进行傅立叶变换之前，将采集的自由感应衰减信号充零至64 K。使用TopSpin 2.1软件对所有核磁共振氢谱进行相位校正和基线调整，以TSP的甲基峰定标(CH
3
, δ0.00)。通过Chenomx NMR套装7.0和参考我们已有的工作
归属代谢物。
为了发掘核磁共振氢谱中包含的所有代谢物信息，将尿液核磁共振氢谱从δ0.5~10.0 ppm分别以0.01 ppm和0.0015 ppm为间隔进行自动分段积分。尿液核磁共振氢谱除去预饱和压水峰后的残留水峰和尿素峰(分别为4.63~5.27 ppm和5.74~5.99 ppm)。为了补偿样本间的浓度差异，对每一段积分值相对于该谱总积分值均进行归一化处理。将0.01 ppm为区间归一化后得到的积分值导入SIMCA-P+12.0软件包(瑞典Umetrics公司)进行模式识别分析。首先对数据进行中心化处理，然后进行偏最小二乘法判别分析(partial least-squares discriminant analysis，PLS-DA)。PLS-DA的第一和第二个主成分代表矩阵中最大的信息变量，其构建的得分图上每一点代表一个特定样本的代谢模式；相应的载荷图上每一点代表了核磁共振谱中的一个积分区间，反映了积分区间对分离不同组别的贡献大小
2
是PLS-DA模型的重要参数，
2
是前两个主成分所包含总信息的百分数，可以反映模型的区分程度，而
2
值则代表模型的预测能力。
将0.0015 ppm为区间归一化后得到的积分值导入SPSS 13.0软件进行统计学分析，计量资料以均数±标准差(
s
)表示，组间比较采用独立样本
t
检验，
P
< 0.05为差异有统计学意义。
是野生型小鼠和
APP/PS1
小鼠尿液的核磁共振氢谱。通过核磁共振氢谱可以同时获得许多小分子代谢物信息，其代谢物归属为2-羟基丁酸、异丁酸、甲基琥珀酸、3-羟基丁酸、苏氨酸、乙酸、琥珀酸、2-酮戊二酸、柠檬酸、甲胺、二甲胺、三甲胺、肌酸、顺乌头酸等。从谱图中可以看出，两组的代谢物浓度具有明显差异，与野生型小鼠比较，
APP/PS1
小鼠尿液的代谢轮廓明显不同。
目前，关于AD的病理机制，包括Aβ的生成和聚合、氧化应激、炎症、Tau蛋白的高度磷酸化及聚集、微血管变化和兴奋性中毒等研究已逐渐深入，发现不少生物标志物
。绝大多数标志物从已确诊的患者或动物模型中获得，但迄今还没有一个可实际应用于AD早期诊断的体液生物学标志物
。在本研究中，我们采用基于核磁共振的代谢组学技术，取经典AD模型
APP/PS1
双转基因小鼠在4月龄时的尿液，从整体代谢水平探索AD发病初期的代谢差异。尿液具有易收集、无损伤、预处理方法简单等优点，可在一定程度上反映机体的功能状态，其特异性的代谢物可作为观察指标
。本研究结果显示，通过查询KEGG数据库
，早期AD模型小鼠尿液中多种代谢物含量发生变化，多条代谢通路受到影响，如
所示。
References
1.
BALLARD C, GAUTHIER S, CORBETT A, et al. Alzheimer's disease.
Lancet.
2011;
377
(9770):1019–1031. doi: 10.1016/S0140-6736(10)61349-9.
[BALLARD C, GAUTHIER S, CORBETT A, et al. Alzheimer's disease[J]. Lancet, 2011, 377(9770):1019-1031.]
[
PubMed
] [
CrossRef
]
[
Google Scholar
]
2.
PRINCE M, COMAS-HERRERA A, KNAPP M, et al. World Alzheimer report 2016[EB/OL].[2018-07-25]. .
3.
DONG M J, PENG B, LIN X T, et al. The prevalence of dementia in the People's Republic of China:a systematic analysis of 1980-2004 studies.
Age Ageing.
2007;
36
(6):619–624. doi: 10.1093/ageing/afm128.
[DONG M J, PENG B, LIN X T, et al. The prevalence of dementia in the People's Republic of China:a systematic analysis of 1980-2004 studies[J]. Age Ageing, 2007, 36(6):619-624.]
[
PubMed
] [
CrossRef
]
[
Google Scholar
]
4.
唐 毅. 阿尔茨海默病诊断标准:从临床诊断到病理生理诊断
山东大学学报(医学版)
2017;
55
(10):14–20. doi: 10.6040/j.issn.1671-7554.0.2017.320.
[唐毅.阿尔茨海默病诊断标准:从临床诊断到病理生理诊断[J].山东大学学报(医学版), 2017, 55(10):14-20.]
[
CrossRef
]
[
Google Scholar
]
6.
FUKUHARA K, OHNO A, OTA Y, et al. NMR-based metabolomics of urine in a mouse model of Alzheimer's disease:identification of oxidative stress biomarkers.
J Clin Biochem Nutr.
2013;
52
(2):133–138. doi: 10.3164/jcbn.12-118.
[FUKUHARA K, OHNO A, OTA Y, et al. NMR-based metabolomics of urine in a mouse model of Alzheimer's disease:identification of oxidative stress biomarkers[J]. J Clin Biochem Nutr, 2013, 52(2):133-138.]
[
PMC free article
]
[
PubMed
] [
CrossRef
]
[
Google Scholar
]
8.
LEWIS J, DICKSON D W, LIN W L, et al. Enhanced neurofibrillary degeneration in transgenic mice expressing mutant tau and APP.
Science.
2001;
293
(5534):1487–1491. doi: 10.1126/science.1058189.
[LEWIS J, DICKSON D W, LIN W L, et al. Enhanced neurofibrillary degeneration in transgenic mice expressing mutant tau and APP[J]. Science, 2001, 293(5534):1487-1491.]
[
PubMed
] [
CrossRef
]
[
Google Scholar
]
9.
LALONDE R, KIM H D, FUKUCHI K. Exploratory activity, anxiety, and motor coordination in bigenic
APP
swe +
PS1
/△E9 mice
.
Neurosci Lett.
2004;
369
(2):156–161. doi: 10.1016/j.neulet.2004.07.069.
[LALONDE R, KIM H D, FUKUCHI K. Exploratory activity, anxiety, and motor coordination in bigenic
APP
swe +
PS1
/△E9 mice[J]. Neurosci Lett, 2004, 369(2):156-161.
]
[
PubMed
] [
CrossRef
]
[
Google Scholar
]
10.
VOLIANSKIS A, KØSTNER R, MØLGAARD M, et al. Episodic memory deficits are not related to altered glutamatergic synaptic transmission and plasticity in the CA1 hippocampus of the
APP
swe/
PS1
δE9-deleted transgenic mice model of ß-amyloidosis
.
Neurobiol Aging.
2010;
31
(7):1173–1187. doi: 10.1016/j.neurobiolaging.2008.08.005.
[VOLIANSKIS A, KØSTNER R, MØLGAARD M, et al. Episodic memory deficits are not related to altered glutamatergic synaptic transmission and plasticity in the CA1 hippocampus of the
APP
swe/
PS1
δE9-deleted transgenic mice model of ß-amyloidosis[J]. Neurobiol Aging, 2010, 31(7):1173-1187.
]
[
PubMed
] [
CrossRef
]
[
Google Scholar
]
11.
National Research Council (US) Committee .
Guide for the Care and Use of Laboratory Animals.
Washington DC: National Academies Press; 2011.
[National Research Council (US) Committee. Guide for the Care and Use of Laboratory Animals[M]. Washington DC:National Academies Press, 2011.]
[
Google Scholar
]
13.
ZHAO X, FRITSCHE J, WANG J, et al. Metabonomic fingerprints of fasting plasma and spot urine reveal human pre-diabetic metabolic traits.
Metabolomics.
2010;
6
(3):362–374. doi: 10.1007/s11306-010-0203-1.
[ZHAO X, FRITSCHE J, WANG J, et al. Metabonomic fingerprints of fasting plasma and spot urine reveal human pre-diabetic metabolic traits[J]. Metabolomics, 2010, 6(3):362-374.]
[
PMC free article
]
[
PubMed
] [
CrossRef
]
[
Google Scholar
]
15.
TARAWNEH R, HOLTZMAN D M. Biomarkers in translational research of Alzheimer's disease.
Neuropharmacology.
2010;
59
(4-5):310–322. doi: 10.1016/j.neuropharm.2010.04.006.
[TARAWNEH R, HOLTZMAN D M. Biomarkers in translational research of Alzheimer's disease[J]. Neuropharmacology, 2010, 59(4-5):310-322.]
[
PMC free article
]
[
PubMed
] [
CrossRef
]
[
Google Scholar
]
18.
KANEHISA M, FURUMICHI M, TANABE M, et al. KEGG:new perspectives on genomes, pathways, diseases and drugs.
Nucleic Acids Res.
2017;
45
(D1):D353–D361. doi: 10.1093/nar/gkw1092.
[KANEHISA M, FURUMICHI M, TANABE M, et al. KEGG:new perspectives on genomes, pathways, diseases and drugs[J]. Nucleic Acids Res, 2017, 45(D1):D353-D361.]
[
PMC free article
]
[
PubMed
] [
CrossRef
]
[
Google Scholar
]
19.
GORMAN A M, CECCATELLI S, ORRENIUS S. Role of mitochondria in neuronal apoptosis.
Dev Neurosci.
2000;
22
(5-6):348–358. doi: 10.1159/000017460.
[GORMAN A M, CECCATELLI S, ORRENIUS S. Role of mitochondria in neuronal apoptosis[J]. Dev Neurosci, 2000, 22(5-6):348-358.]
[
PubMed
] [
CrossRef
]
[
Google Scholar
]
20.
KIRKLAND R A, FRANKLIN J L. Bax and caspases regulate increased production of mitochondria-derived reactive species in neuronal apoptosis:lack of a role for depletion of cytochrome c from the mitochondrial electron transport chain.
http://pubmedcentralcanada.ca/pmcc/articles/PMC5668918/
Biochem Biophys Rep.
2015;
4
:158–168.
[KIRKLAND R A, FRANKLIN J L. Bax and caspases regulate increased production of mitochondria-derived reactive species in neuronal apoptosis:lack of a role for depletion of cytochrome c from the mitochondrial electron transport chain[J]. Biochem Biophys Rep, 2015, 4:158-168.]
[
PMC free article
]
[
PubMed
]
[
Google Scholar
]
21.
WU H, ZHANG X, LIAO P, et al. NMR spectroscopic-based metabonomic investigation on the acute biochemical effects induced by Ce(NO3)3 in rats.
J Inorg Biochem.
2005;
99
(11):2151–2160. doi: 10.1016/j.jinorgbio.2005.07.014.
[WU H, ZHANG X, LIAO P, et al. NMR spectroscopic-based metabonomic investigation on the acute biochemical effects induced by Ce(NO3)3 in rats[J]. J Inorg Biochem, 2005, 99(11):2151-2160.]
[
PubMed
] [
CrossRef
]
[
Google Scholar
]
22.
BUCKLEY B M, WILLIAMSON D H. Origins of blood acetate in the rat.
Biochem J.
1977;
166
(3):539–545. doi: 10.1042/bj1660539.
[BUCKLEY B M, WILLIAMSON D H. Origins of blood acetate in the rat[J]. Biochem J, 1977, 166(3):539-545.]
[
PMC free article
]
[
PubMed
] [
CrossRef
]
[
Google Scholar
]
24.
VIDONI E D, TOWNLEY R A, Honea R A, et al. Alzheimer disease biomarkers are associated with body mass index.
Neurology.
2011;
77
(21):1913–1920. doi: 10.1212/WNL.0b013e318238eec1.
[VIDONI E D, TOWNLEY R A, Honea R A, et al. Alzheimer disease biomarkers are associated with body mass index[J]. Neurology, 2011, 77(21):1913-1920.]
[
PMC free article
]
[
PubMed
] [
CrossRef
]
[
Google Scholar
]
25.
HOYER S. Glucose metabolism and insulin receptor signal transduction in Alzheimer disease.
Eur J Pharmacol.
2004;
490
(1-3):115–125. doi: 10.1016/j.ejphar.2004.02.049.
[HOYER S. Glucose metabolism and insulin receptor signal transduction in Alzheimer disease[J]. Eur J Pharmacol, 2004, 490(1-3):115-125.]
[
PubMed
] [
CrossRef
]
[
Google Scholar
]
26.
PELLICCIARI A, POSAR A, CREMONINI M A, et al. Epilepsy and trimethylaminuria:a new case report and literature review.
Brain Dev.
2011;
33
(7):593–596. doi: 10.1016/j.braindev.2010.09.007.
[PELLICCIARI A, POSAR A, CREMONINI M A, et al. Epilepsy and trimethylaminuria:a new case report and literature review[J]. Brain Dev, 2011, 33(7):593-596.]
[
PubMed
] [
CrossRef
]
[
Google Scholar
]
28.
PSIHOGIOS N G, GAZI I F, ELISAF M S, et al. Gender-related and age-related urinalysis of healthy subjects by NMR-based metabonomics.
NMR Biomed.
2008;
21
(3):195–207. doi: 10.1002/(ISSN)1099-1492.
[PSIHOGIOS N G, GAZI I F, ELISAF M S, et al. Gender-related and age-related urinalysis of healthy subjects by NMR-based metabonomics[J]. NMR Biomed, 2008, 21(3):195-207.]
[
PubMed
] [
CrossRef
]
[
Google Scholar
]
30.
MINTER M R, ZHANG C, LEONE V, et al. Antibiotic-induced perturbations in gut microbial diversity influences neuro-inflammation and amyloidosis in a murine model of Alzheimer's disease.
Sci Rep.
2016;
6
:30028. doi: 10.1038/srep30028.
[MINTER M R, ZHANG C, LEONE V, et al. Antibiotic-induced perturbations in gut microbial diversity influences neuro-inflammation and amyloidosis in a murine model of Alzheimer's disease[J]. Sci Rep, 2016, 6:30028.]
[
PMC free article
]
[
PubMed
] [
CrossRef
]
[
Google Scholar
]
31.
MINTER MR, HINTERLEITNER R, MEISEL M, et al. Antibiotic-induced perturbations in microbial diversity during post-natal development alters amyloid pathology in an aged APPSWE/PS1△E9 murine model of Alzheimer's disease.
Sci Rep.
2017;
7
(1):10411. doi: 10.1038/s41598-017-11047-w.
[MINTER MR, HINTERLEITNER R, MEISEL M, et al. Antibiotic-induced perturbations in microbial diversity during post-natal development alters amyloid pathology in an aged APPSWE/PS1△E9 murine model of Alzheimer's disease[J]. Sci Rep, 2017, 7(1):10411.]
[
PMC free article
]
[
PubMed
] [
CrossRef
]
[
Google Scholar
]
32.
ZHOU J, CHAN L, ZHOU S. Trigonelline:a plant alkaloid with therapeutic potential for diabetes and central nervous system disease.
Curr Med Chem.
2012;
19
(21):3523–3531. doi: 10.2174/092986712801323171.
[ZHOU J, CHAN L, ZHOU S. Trigonelline:a plant alkaloid with therapeutic potential for diabetes and central nervous system disease[J]. Curr Med Chem, 2012, 19(21):3523-3531.]
[
PubMed
] [
CrossRef
]
[
Google Scholar
]
Articles from
Journal of Zhejiang University (Medical Sciences)
are provided here courtesy of
Zhejiang University Press
3,
*
