CN113960199B - 血浆代谢物在诊断帕金森病严重程度方面的应用 - Google Patents
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Abstract
本发明公开了血浆代谢物在诊断帕金森病严重程度方面的应用，具体是在制备诊断帕金森病严重程度的检测试剂或检测物中的应用，其中血浆代谢物包括血浆支链氨基酸和/或芳香族氨基酸，支链氨基酸选自亮氨酸、异亮氨酸和缬氨酸中的一种或三种组合，芳香族氨基酸为酪氨酸和/或苯丙氨酸。本发明以血浆为样本，生物样本易得，收集较简便、数量大，价格低廉、检测周期短，通过检测血浆标志物可以判断PD疾病严重程度，是一种可靠、高效、便捷的新方法，用于PD患者的治疗和预后具有广阔的应用前景。
Description
技术领域
本发明属于生物医学领域，具体涉及血浆代谢物在诊断帕金森病严重程度方面的应用。
背景技术
帕金森病(Parkinson's disease,PD)是一种常见的中老年神经系统退行性疾病，主要病理改变为黑质多巴胺能神经元进行性退变和路易小体形成，以静止性震颤、肌强直、运动迟缓、姿势平衡障碍等运动症状和嗅觉减退、便秘、睡眠行为异常和抑郁等非运动症状为显著特征。PD患者症状会逐渐加重，评估症状的严重程度对于制定合理的治疗方案以及判断疾病预后至关重要。
目前已有的关于PD疾病严重程度的生物学标志物研究主要集中在临床症状、生物化学、影像及便携设备几方面，但是均存在以下不足：
(1)临床症状：通过国际运动障碍学会统一帕金森病评定量表(MDS-UPDRS)进行临床评估，但该评估需要到医疗机构由专业医生进行评估，评估过程耗时较长，评估结果容易受到评估人员主观因素的影响。
(2)生物化学：PD患者血浆载脂蛋白A1(ApoA1)水平同对照者相比明显降低(Swanson,C.R.et al.Lower plasma apolipoprotein A1 levels are found inParkinson’s disease and associate with apolipoprotein A1genotype.Mov.Disord.30,805–812(2015).)。更为重要的是，在早期未经药物治疗的PD患者中，血浆ApoA1水平越低，疾病严重程度越高(Swanson,C.R.et al.Plasmaapolipoprotein A1 associates with age at onset and motor severity in earlyParkinson’s disease patients.Mov.Disord.30,1648–1656(2015).)，提示血浆ApoA1可能作为预测PD疾病严重程度的生物标志物，但该生物标志物的诊断价值尚不明确，不能用于PD患者。
(3)影像：Tomoyoshi Kuribara等发现，PD患者进行DaTSCAN注射后，通过SPECT/CT扫描，进行多巴胺转运体(DAT)成像，其中纹状体的特异性结合率(SBR)与MDS-UPDRS Part3评分呈显著负相关，提示DAT成像有望成为预测PD疾病严重程度的影像学生物标志物(Kuribara,T.et al.Neuroimaging and neurophysiological evaluation of severityof Parkinson’s disease.J.Clin.Neurosci.74,135–140(2020).)。Beatriz Jimenez等人发现，光学相干断层扫描(OCT)测量的视盘周围视网膜神经纤维层(RNFL)厚度的减少程度与PD患者UPDRS评分增加有关，提示RNFL厚度有望成为判断PD患者疾病严重程度的生物标志物(Jiménez,B.,Ascaso,F.J.,Cristóbal,J.A.&López del Val,J.Development of aprediction formula of Parkinson disease severity by optical coherencetomography.Mov.Disord.29,68–74(2014).)，但以上生物标志物的是否能够判断PD患者疾病严重程度尚需进一步研究。
(4)便携设备：MeysamAsgari等人开发一种可以居家远程监测的工具，该工具通过收集患者语言发音，分析其特征(例如语速、音调、谐波噪声比等)，利用特定的算法处理进行疾病严重程度的判断(Asgari,M.&Shafran,I.Predicting severity of Parkinson’sdisease from speech.2010Annu.Int.Conf.IEEE Eng.Med.Biol.Soc.2010,5201–5204(2010).)，该方法尚处于实验阶段，是否能够投入临床应用还不明确。
因此，需要找到一个敏感度和特异性高，可在临床广泛应用且能够客观、快速、高效反映PD疾病严重程度的诊断生物学标记物。
近年来，研究证实PD患者存在肠道菌群紊乱，肠道菌群对于膳食营养物质的代谢和吸收有很大影响，而氨基酸的吸收主要通过饮食摄入，肠道菌群紊乱可能会影响PD患者体内氨基酸的含量。研究通过代谢组学方法发现PD患者的血浆氨基酸浓度较健康对照组发生显著变化(Shao,Y.et al.Comprehensive metabolic profiling of Parkinson’sdisease by liquid chromatography-mass spectrometry.Mol.Neurodegener.16,1–15(2021).)。由于肠道菌群与PD疾病严重程度有关，随着疾病严重程度变化，血浆氨基酸含量也可能会发生变化，PD患者能量消耗增加，能够增加血浆氨基酸的消耗。另外，PD患者合并胃肠道功能障碍，能够影响氨基酸的吸收，随着PD疾病严重程度增加，胃肠功能障碍加剧，会进一步影响氨基酸的吸收，由此推测血浆氨基酸含量可能与PD疾病严重程度相关。
支链氨基酸(BCAAs)包括亮氨酸(Leu)、异亮氨酸(Ile)和缬氨酸(Val)，是人体必需氨基酸，而且在大脑中充当氮供体，维持星形胶质细胞和神经元之间的谷氨酸-谷氨酰胺循环。BCAAs可通过激活谷氨酸脱氢酶促进谷氨酸的分解代谢，还能够通过转氨作用产生支链酮酸，对毒性水平的谷氨酸产生“缓冲效应”，由于谷氨酸通过其兴奋毒性、氧化应激和免疫兴奋毒性与帕金森病的发生和发展密切相关，因此，BCAAs可能通过调节谷氨酸代谢在PD中发挥保护作用。酪氨酸(Tyr)和苯丙氨酸(Phe)是芳香族氨基酸(AAAs)，是产生多巴胺的关键底物，多巴胺是帕金森病中缺乏的神经递质，推断可以通过检测PD患者血浆BCAAs和AAAs浓度，分析血浆BCAAs和AAAs浓度与PD疾病严重程度的关系，明确血浆BCAAs和AAAs浓度是否能够预测PD疾病严重程度。
发明内容
基于此，本发明的目的是提供一种血浆代谢物在制备诊断帕金森病严重程度的检测试剂或检测物中的应用，通过收集PD患者临床资料，基于高效液相色谱-荧光检测法(HPLC-FLD)对PD患者血浆BCAAs及AAAs进行分析，发现血浆BCAAs及AAAs浓度与PD疾病严重程度呈显著负相关，首次筛选出可作为诊断PD疾病严重程度的血浆标志物BCAAs及AAAs。
为实现上述目的，本发明采用的技术方案是：
本发明提供血浆代谢物在制备诊断帕金森病严重程度的检测试剂或检测物中的应用，所述血浆代谢物包括血浆支链氨基酸和/或芳香族氨基酸。
优选地，所述支链氨基酸选自Leu、Ile和Val中的一种或三种联合，所述芳香族氨基酸为Tyr和/或Phe。
更优选地，所述血浆代谢物为Leu，且Leu的判断临界浓度为1.265μg/mL。优选地，所述检测试剂为血液检测试剂。
优选地，所述检测物为试剂盒。
更优选地，所述试剂盒包含所述血浆代谢物的标准化学品，分别对受试者血浆中所述血浆代谢物进行定性。
与现有技术相比，本发明的有益效果在于：
(1)本发明首次在中国PD患者中检测血浆BCAA及AAA浓度，发现血浆BCAA及AAA浓度与PD患者疾病严重程度呈显著负相关，进一步发现中晚期PD患者血浆BCAA及AAA浓度较早期PD显著下降。通过ROC分析发现，单独及联合氨基酸能够预测PD疾病严重程度，特别是血Leu的浓度预测PD疾病严重程度具有较高的灵敏度和特异度。
(2)本发明以血浆为样本，生物样本易得，收集较简便、数量大，价格低廉、检测周期短，通过检测血浆标志物就可以客观地判断PD疾病严重程度，是一种可靠、高效、便捷的新方法，用于PD患者的治疗和预后具有广阔的应用前景和潜能，特别适用于我国人群中大范围临床诊断PD疾病严重程度。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点会变得更显著：
图1是PD患者血浆BCAAs及AAAs浓度与疾病严重程度相关性分析。
图2是血浆BCAA及AAA浓度单独预测PD疾病严重程度。
图3是血浆BCAA和AAA联合预测PD疾病严重程度。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面结合本发明实施例的附图，对本发明实施例的技术方案进行清楚、完整地描述。显然，所描述的实施例是本发明的一部分实施例，而不是全部的实施例。基于所描述的本发明的实施例，本领域普通技术人员在无需创造性劳动的前提下所获得的所有其它实施例，都属于本发明保护的范围。
实施例1
以下通过临床试验验证血浆BCAAs及AAAs浓度与PD疾病严重程度的相关性，包括以下几个步骤：1)收集PD患者临床资料；2)检测PD患者血浆BCAAs及AAAs水平；3)分析PD患者血浆BCAAs及AAAs与临床资料的相关性；4)根据血浆BCAAs及AAAs判断PD严重程度。本临床试验得到上海交通大学医学附属瑞金医院伦理委员会批准。所有受试者自愿参加，均被口头告知实验目的并签署知情同意书。
(1)收集PD患者临床资料
从门诊收集PD患者及健康对照者。PD患者入组标准包括临床确诊的原发性帕金森病患者，诊断标准参照英国脑库帕金森病诊断标准。排除标准包括：(1)非典型或继发性帕金森病，(2)严重慢性疾病(如糖尿病、心力衰竭、肝硬化、恶性肿瘤、血液病或自身免疫性疾病或炎症性胃肠疾病)，(3)入组前3个月内使用益生菌或抗生素补充剂；健康对照入组要求无疾病史。
临床资料的收集采取面对面访谈的形式，疾病严重程度在“开”期通过H&Y分期和MDS-UPDRS量表进行评估，PD疾病严重程度通过H&Y分期进行分类：早期PD：H&Y分期<2.5；中晚期PD：H&Y分期≥2.5。
禁食至少10小时后，采集106个PD患者静脉血样本，并在4℃离心分离获得血浆，储存于-80℃冰箱。
(2)检测PD患者血浆BCAAs及AAAs水平
采用HPLC-FLD检测血浆BCAAs及AAAs，具体如下：
样品预处理：取200ul血浆样本，加入4倍体积的乙醇，充分混合，置4℃冰箱静置30分钟，然后在0℃下13000r离心25分钟，0.45um滤膜过滤后，氮气吹干，用100ul 0.1M HCL复溶，取10ul样本溶液，加入25ul硼酸盐缓冲液，加入衍生化试剂，充分斡旋，加入200ul的稀释剂斡旋2分钟后进样。
液相条件：流动相为A：10mM磷酸氢二钠，10mM硼酸钠，B：甲醇：乙腈：水(45:45:10)。色谱柱为：YMC-C18-EXRS(150mm×4.6mm,3μm)。进样器温度：4℃；进样量：10μL；流速：1mL min-1。紫外波长338nm和236nm，激发波长是260nm，发射波长是325nm。
(3)分析PD患者血浆BCAAs及AAAs与临床资料的相关性
分析PD患者血浆BCAAs及AAAs浓度与疾病严重程度的相关性(图1)，比较早期及中晚期PD患者血浆BCAAs及AAAs浓度(表1)，发现血浆BCAA及AAA浓度与PD患者疾病严重程度呈显著负相关，中晚期PD患者血浆BCAA及AAA浓度较早期PD显著下降。
表1
另外，用ROC曲线进一步区分早期PD与中晚期PD，结果如下：
通过血浆BCAAs及AAAs中每种氨基酸单独预测PD疾病严重程度(图2)，Leu单独预测，曲线下面积(AUC)为0.71，灵敏度为74.1％，特异度为62.5％；Ile单独预测，AUC为0.68，灵敏度为60.3％，特异度为58.3％；Val单独预测，AUC为0.70，灵敏度为65.5％，特异度为60.4％；Phe单独预测，AUC为0.70，灵敏度为67.2％，特异度为66.7％；Tyr单独预测，AUC为0.61，灵敏度为60.3％，特异度为62.5％。
通过血浆BCAA和AAA联合预测PD疾病严重程度(图3)。BCAA预测，AUC为0.72，灵敏度为70.7％，特异度为62.5％；AAA预测，AUC为0.70，灵敏度为70.7％，特异度为62.5％；BCAA+AAA预测，AUC为0.73，灵敏度为65.5％，特异度为60.4％。综上所述，血浆Leu的浓度作为生物标志物预测PD疾病严重程度具有较高的灵敏度和特异度。
(4)根据上述早期PD及中晚期PD血浆BCAA及AAA浓度判定PD疾病严重程度。例如，当亮氨酸浓度低于1.265μg/mL时，指示受试者为中晚期PD。
综上所述，本发明以血浆为样本，生物样本易得，收集较简便、数量大，价格低廉、检测周期短，通过HPLC-FLD检测血浆标志物就可以判断PD疾病严重程度，是一种可靠、高效、便捷的新方法，对PD患者的治疗、预后具有广阔的应用前景和潜能，适合用于我国人群中大范围临床诊断PD疾病严重程度。
Claims (1)
1.血浆代谢物在制备诊断帕金森病严重程度的检测试剂或检测物中的应用，所述血浆代谢物为亮氨酸，且亮氨酸的判断临界浓度为1.265μg/mL；
当亮氨酸浓度低于1.265μg/mL时，指示受试者为中晚期帕金森病患者；
所述检测试剂为血液检测试剂；
所述检测物为试剂盒，含所述血浆代谢物的标准化学品，分别对受试者血浆中所述血浆代谢物进行定性。
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