CN111175398A - 一种用于诊断痛风的试剂盒及其应用 - Google Patents
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Abstract
一种用于诊断痛风的试剂盒及其应用，属于医疗机械领域。本发明提供的技术方案如下：一种用于诊断痛风的试剂盒，所述的试剂盒中含有用于检测样本中的焦谷氨酸、2‑甲基丁酰肉碱和Phe‑Phe含量的标准品。本发明通过LC‑MS鉴定了痛风的血清和尿液代谢组学特征，发现了三种代谢物，即焦谷氨酸(pyroglutamic acid and)、2‑甲基丁酰肉碱(2‑methylbutyryl carnitine)和Phe‑Phe，它们可以将痛风患者从健康对照中分离出来，并作为早期诊断的有效手段，操作简单，确诊率高。
Description
技术领域
本发明涉及一种用于诊断痛风的试剂盒及其应用，属于医疗机械领域。
技术背景
痛风是一种常见的以单钠尿酸盐沉积在关节炎滑液和组织为特征的炎性疾病，过去几十年来，全世界痛风发病率持续增长，目前广泛应用的痛风临床评价和诊断标准是基于外周关节表现与血尿酸浓度。然而，对于一些患者，血清尿酸浓度可以是正常的，即使在关节炎发作时。此外，血清尿酸水平受饮食影响特别敏感，关节炎的发作可能是暂时性的，而且往往不经治疗自发缓解，它很容易被病人忽略。痛风早期症状轻微，尿酸正常的患者经常延迟诊断，迫切需要预测新的生物标记物以便更好地诊断痛风。
代谢物是代谢的底物和产物，它驱动着细胞的基本功能并表示基因组的下游输出和来自环境。代谢组学，其中小分子代谢物(代谢组)被广泛认为是最接近于表型，由于代谢组学的固有敏感性，甚至在途径上的细微变化可在代谢物中检测到，是发现生物标志物的理想工具。另外，通过代谢组学揭示了潜在的代谢途径，具有提供洞察力的潜力在疾病机理上创新发展信息与分析技术。代谢物组学是系统地覆盖关键代谢物的低分子量化合物的综合定量测量，其代表中间代谢的整个途径。在系统生物学方法中，它提供了由遗传蓝图，调节，蛋白质丰度和修饰以及环境影响决定的变化的功能读数。分析大的阵列的代谢物的能力提取反映明显生物事件真实功能终点的生物化学信息，而其他功能基因组学技术如转录组学和蛋白质组学虽然非常有价值，但仅仅表明了表型反应的潜在原因。因此，除非添加功能验证，否则它们不一定能预测表型水平的疾病状态。代谢物组学通过特别描述这种功能信息来弥合这种信息沟，因为生物流体和组织中的代谢物差异提供了与各种表型反应的最密切联系。表型不一定通过基因型预测，基因型和表型之间的沟由许多生化反应所跨越，每个反应对各种影响具有个体依赖性，所述影响包括药物，营养和环境因素，在从基因到表型的这种生物分子链中，代谢物是与表型最密切相关的可量化分子。
发明内容
为克服上述提到的问题，本发明使用了一种更灵敏的代谢组学平台-超高效液相色谱-质谱(UPLC-MS)，在痛风患者和健康对照两个队列中进行代谢组学分析，研究的目的是揭示痛风患者血清和尿液的代谢改变，并用发现队列建立了诊断模型。
本发明提供的技术方案如下：
一种用于诊断痛风的试剂盒，所述的试剂盒中含有用于检测样本中的焦谷氨酸、2-甲基丁酰肉碱和Phe-Phe含量的标准品。
优选的，所述的试剂盒中还含有检测如下任何一种物质的标准品：焦谷氨酸(pyroglutamic acid)，Trp-Phe，黄嘌呤(xanthine)，阿拉伯酰基次黄嘌呤(arabinosylhypoxanthine)，2-甲基丁酰基肉碱(2-methylbutyrylcarnitine),16(R)-HETE,icomucret,LysoPE(0:0/20:2(11Z,14Z)，丙酸睾酮(testosterone propionate)。
优选的，所述的方法包括有：测定患者样本中的标志物的含量，然后将该含量和正常对照组中的所述生物标志物的量进行对比，所述的生物标志物为焦谷氨酸、2-甲基丁酰肉碱和Phe-Phe。
优选的，所述的生物标志物还包括有以下任何一种：焦谷氨酸(pyroglutamicacid)，Trp-Phe，黄嘌呤(xanthine)，阿拉伯酰基次黄嘌呤(arabinosylhypoxanthine)，2-甲基丁酰基肉碱(2-methylbutyrylcarnitine),16(R)-HETE,icomucret,LysoPE(0:0/20:2(11Z,14Z)，丙酸睾酮(testosterone propionate)。
本发明还提供一种生物标志物或特异性结合该生物标志物的抗体或抗体片段在制备1)用于诊断患者中的痛风或者评价患者患痛风的风险；或2)用于治疗痛风的药物中的用途，所述的生物标志物为焦谷氨酸、2-甲基丁酰肉碱和Phe-Phe。
进一步的，所述的生物标志物还包括有以下任何一种：焦谷氨酸(pyroglutamicacid)，Trp-Phe，黄嘌呤(xanthine)，阿拉伯酰基次黄嘌呤(arabinosylhypoxanthine)，2-甲基丁酰基肉碱(2-methylbutyrylcarnitine),16(R)-HETE,icomucret,LysoPE(0:0/20:2(11Z,14Z)，丙酸睾酮(testosterone propionate)。
本发明通过LC-MS鉴定了痛风的血清和尿液代谢组学特征，发现了三种代谢物，即焦谷氨酸(pyroglutamic acid and)、2-甲基丁酰肉碱(,2-methylbutyryl carnitine)和Phe-Phe，它们可以将痛风患者从健康对照中分离出来，并作为早期诊断的有效手段，操作简单，确诊率高。
附图说明
图1为发现队列中40种差异代谢物的分类表现：(a)基于40种代谢物PCA分析的散点图；(b)基于40种代谢物的OPLS-DA分析的散点图；(c)40种代谢物诊断痛风的ROC曲线分析；(d)每种代谢物的聚类分析热图。
图2为8种常见重叠代谢物在发现队列(a-d)和验证队列(e-h)分类中的表现：(a，f)基于8种代谢物的主成分分析散点图；(b，g)8种代谢物的OPLS-DA散点图；(c，h)8种代谢物诊断痛风的ROC分析；(d，e)每种代谢物的聚类分析热图。
具体实施方式
申请人选取30名痛风患者作为实验组，30名经检查未患痛风者作为对照组，基本情况如下：
然后对抽取的样本进行如下的处理：
(一)样本预处理
1.样本置于室温解冻，用移液枪吸取100μL血清样本于1.5mL EP管中。
2.加入300μL甲醇以沉淀血清中蛋白，并加入10μL内标(2.9mg/mL，2-氯苯丙氨酸)。
3.涡旋混匀30s，而后置于4℃离心机中，12000rpm离心15min。
4.吸取200μL上清，转入进样小瓶中待检测。
(二)UPLC/MS分析
1.仪器分析平台：UPLC-MS(Thermo,Ultimate 3000LC,Q Exactive)
2.色谱柱：C18色谱柱Hypersil GOLD(100×2.1mm,1.9μm)
3.色谱分离条件为：柱温为40℃；流速0.3mL/min；
流动相组成A：水+0.1％甲酸，B：乙腈+0.1％甲酸；
进样量为4μL，自动进样器温度4℃。
4.流动相梯度洗脱程序见Table 1：
Table 1.流动相洗脱程序
5.质谱检测参数：
正模式：加热器温度300℃；鞘气流速：45arb；辅助气流速：15arb；尾气流速：1arb；电喷雾电压：3.0KV；毛细管温度：350℃；S-Lens RF Level,30％。
负模式：加热器温度300℃；鞘气流速：45arb；辅助气流速：15arb；尾气流速：1arb；电喷雾电压：3.2KV；毛细管温度：350℃；S-Lens RF Level,60％。
(三)数据分析
使用Compoud Discoverer软件(Thermo公司)对LC/MS检测数据进行提取和预处理，并在Excel 2010中对数据进行归一化及后期编辑，最后整理成二维数据矩阵形式，包含保留时间(RT(Retention time))、分子量(CompMW)、观察量(样本名称)、可提取物质个数(ID)和峰强度等信息。将编辑后的数据矩阵导入SIMCA-P 13.0(Umetrics AB，Umea，Sweden)软件进行多元统计分析，得到的结果如下：
附表1：Discovery队列中痛风患者与健康对照的40种差异血清代谢物
从附表1中可以看出，血清中40种代谢物(见附表1)的浓度，主要包括嘌呤衍生物、氨基酸、脂肪酰基等，显示出痛风患者和健康对照之间的显着差异。其中9种代谢物在痛风诊断中的AUC大于尿酸，表明这些代谢物可以作为新的生物标志物，9种代谢物如下：焦谷氨酸(pyroglutamic acid)，Trp-Phe，黄嘌呤(xanthine)，阿拉伯酰基次黄嘌呤(arabinosylhypoxanthine)，2-甲基丁酰基肉碱(2-methylbutyrylcarnitine),16(R)-HETE,icomucret,LysoPE(0:0/20:2(11Z,14Z)，丙酸睾酮(testosterone propionate)。
基于上述的结果，发明人勾画了痛风患者尿液代谢组学的轮廓，构建了一个包含40个样本的诊断模型。PCA，OPLS-DA，聚类和ROC分析表明，基于LC-MS的代谢组学是发现痛风生物标志物的一种有前途的技术(参照附图1)。
此外，在另一个队列中进行了初始模型的验证和优化。值得注意的是，有8种代谢物重叠。通过回归分析，8种代谢物中的3种，即焦谷氨酸(pyroglutamicacid)、2-甲基丁酰肉碱(,2-methylbutyryl carnitine)和Phe-Phe具有最佳诊断能力(见附表2)，用这3种代谢物构建的最终模型产生了高AUC值(0.956)，并显着改善了痛风的诊断性能。其中图1为发现队列中40种差异代谢物的分类表现：(a)基于40种代谢物PCA分析的散点图；(b)基于40种代谢物的OPLS-DA分分析的散点图。(c)40种代谢物诊断痛风的ROC曲线分析。(d)每种代谢物的聚类分析热图。
为了进一步对上述的结果进行验证，发明人重新组织了验证队列(实验组和对照组各50)，情况如下：
对样本的处理方式和上述发现队列一样，其结果如下：
验证队列中有8种代谢产物得到验证，包括次黄嘌呤，黄嘌呤，缬氨酸，苯丙氨酸，苯丙氨酸，亮氨酸，焦谷氨酸酸和2-甲基丁酰基肉碱。如附图2所示，代谢产物的变化方向与发现队列中观察到的方向一致。
发明人对两个队列中的8种重复代谢物的Logistic回归分析，结果如下：
OR,odds ratio；CI,confidence interval.
两个队列有8种代谢物重叠。通过回归分析，8种代谢物中的3种，即焦谷氨酸(pyroglutamic acid)2-甲基丁酰肉碱(2-methylbutyryl carnitine)和Phe-Phe，可以将痛风患者从健康对照中分离出来，并作为早期诊断的有效手段。
Claims (6)
1.一种用于诊断痛风的试剂盒，其特征在于，所述的试剂盒中含有用于检测样本中的焦谷氨酸、2-甲基丁酰肉碱和Phe-Phe含量的标准品。
2.如权利要求1所述的用于诊断痛风的试剂盒，其特征在于，所述的试剂盒中还含有检测如下任何一种物质的标准品：Trp-Phe，黄嘌呤，阿拉伯酰基次黄嘌呤，16(R)-HETE，icomucret,LysoPE(0:0/20:2(11Z,14Z)和/或丙酸睾酮。
3.能够结合或者特异性识别生物标志物的配体在制备试剂盒中的应用，所述的试剂盒用于一种诊断患者中的痛风的方法，其特征在于，所述的方法包括有：测定患者样本中的标志物的含量，然后将该含量和正常对照组中的所述生物标志物的量进行对比，所述的生物标志物为焦谷氨酸、2-甲基丁酰肉碱和Phe-Phe。
4.如权利要求1所述的能够结合或者特异性识别生物标志物的配体在制备试剂盒中的应用，其特征在于，所述的生物标志物还包括有以下任何一种：Trp-Phe，黄嘌呤，阿拉伯酰基次黄嘌呤,16(R)-HETE,icomucret,LysoPE(0:0/20:2(11Z,14Z)和丙酸睾酮。
5.生物标志物或特异性结合该生物标志物的抗体或抗体片段在制备1)用于诊断患者中的痛风或者评价患者患痛风的风险；或2)用于治疗痛风的药物中的用途，所述的生物标志物为焦谷氨酸、2-甲基丁酰肉碱和Phe-Phe。
6.如权利要求5所述的用途，其特征在于，所述的生物标志物还包括有以下任何一种：Trp-Phe，黄嘌呤，阿拉伯酰基次黄嘌呤,16(R)-HETE,icomucret,LysoPE(0:0/20:2(11Z,14Z)和丙酸睾酮。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113358758A (zh) * | 2020-03-02 | 2021-09-07 | 中国科学院上海营养与健康研究所 | 痛风诊断生物标志物及其应用 |
| CN116381072A (zh) * | 2023-01-16 | 2023-07-04 | 青岛大学附属医院 | 识别偶发型痛风和频发型痛风的生物标志物及其应用 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007126847A2 (en) * | 2006-03-29 | 2007-11-08 | Merrimack Pharmaceuticals, Inc. | Coadministration of alpha-fetoprotein and a disease modifying anti-rheumatic drug for treating inflammatory arthritic disease |
| US20100035255A1 (en) * | 2008-08-11 | 2010-02-11 | Kaohsiung Medical University | Method and kit for assessing risk of gout and hyperuricemia |
| US20100248253A1 (en) * | 2008-08-11 | 2010-09-30 | Kaohsiung Medical University | Method and kit for assessing risk of gout and hyperuricemia |
| US20170336413A1 (en) * | 2014-10-30 | 2017-11-23 | National Institute Of Advanced Industrial Science And Technology | Rheumatoid arthritis marker |
| CN109001443A (zh) * | 2018-06-19 | 2018-12-14 | 上海市第十人民医院 | 一种用于检测急性痛风发作的早期标志物 |
-
2020
- 2020-01-09 CN CN202010023162.2A patent/CN111175398A/zh active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007126847A2 (en) * | 2006-03-29 | 2007-11-08 | Merrimack Pharmaceuticals, Inc. | Coadministration of alpha-fetoprotein and a disease modifying anti-rheumatic drug for treating inflammatory arthritic disease |
| US20100035255A1 (en) * | 2008-08-11 | 2010-02-11 | Kaohsiung Medical University | Method and kit for assessing risk of gout and hyperuricemia |
| US20100248253A1 (en) * | 2008-08-11 | 2010-09-30 | Kaohsiung Medical University | Method and kit for assessing risk of gout and hyperuricemia |
| US20170336413A1 (en) * | 2014-10-30 | 2017-11-23 | National Institute Of Advanced Industrial Science And Technology | Rheumatoid arthritis marker |
| CN109001443A (zh) * | 2018-06-19 | 2018-12-14 | 上海市第十人民医院 | 一种用于检测急性痛风发作的早期标志物 |
Non-Patent Citations (2)
| Title |
|---|
| 张宏如 等: "针刺代谢组学研究的现状与分析", 《南京中医药大学学报》 * |
| 王凯 等: "氧嗪酸钾诱导高尿酸血症小鼠血清代谢组学研究", 《药学服务与研究》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113358758A (zh) * | 2020-03-02 | 2021-09-07 | 中国科学院上海营养与健康研究所 | 痛风诊断生物标志物及其应用 |
| CN116381072A (zh) * | 2023-01-16 | 2023-07-04 | 青岛大学附属医院 | 识别偶发型痛风和频发型痛风的生物标志物及其应用 |
| CN116381072B (zh) * | 2023-01-16 | 2024-05-31 | 苏州艾麦锶生命科技有限公司 | 识别偶发型痛风和频发型痛风的生物标志物及其应用 |
| WO2024152730A1 (zh) * | 2023-01-16 | 2024-07-25 | 苏州艾麦锶生命科技有限公司 | 识别偶发型痛风和频发型痛风的生物标志物及其应用 |
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