CN112229937B - 用于多囊卵巢综合征诊断的生物标志物和试剂盒及使用方法 - Google Patents
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Abstract
本发明提供了一组可用于检测多囊卵巢综合征不同代谢亚型的生物标志物,并进一步提供包含该等生物标志物的用于检测多囊卵巢综合征的试剂盒及其使用方法,可用于对多囊卵巢综合征的快速高效诊断。
Description
技术领域
本发明属于生物检测技术领域,具体涉及多囊卵巢综合征诊断检测用的生物标志物和试剂盒领域。
背景技术
生育力下降成为严重的社会问题,全球的不孕不育率达到10% -15%,生殖内分泌紊乱是目前导致女性不孕的首位病因。多囊卵巢综合征(polycystic ovary syndrome,PCOS)是育龄妇女常见的内分泌系统疾病,是无排卵性不孕症的最常见原因之一。该疾病最常见的临床表现是女性出现高雄激素血症,导致月经紊乱、稀少、闭经或不规则阴道出血、不孕、肥胖、多毛、子宫内膜过度增生及恶性变化、以及双侧或单侧卵巢呈多囊性改变并多伴有肥胖表型;在糖脂内分泌代谢方面出现胰岛素抵抗,继而引发糖尿病等并发症,远期并发症还包括、心血管疾病、子宫内膜癌等。
传统的多囊卵巢综合征诊断主要依靠月经情况、雄激素水平检测和卵巢B超检测。根据鹿特丹ESHRE/ASRM(2003)标准,须满足下述三项中的两项:月经稀发;临床或者生化雄激素过多症;双侧或单侧卵巢多囊样改变和/或体积增大。但上述方法诊断出的PCOS具有高度异质性,未能引入代谢异常的程度这一重要临床特征对PCOS患者进行亚群的分型。以上问题使得PCOS的诊断与治疗已经成为妇科和内分泌领域共同关注的热点和难点问题。因此,找到一种易于检测的无创性生物标记物诊断方法,结合现有的临床指标,增加代谢特征的指标,来预测和诊断多囊卵巢综合征,对于多囊卵巢综合征的疾病风险评估有很大的意义。目前,市场上还没有针对多囊卵巢综合征的快速高效诊断试剂盒问世,影响到多囊卵巢综合征早期发现及治疗。
肠道微生态主要包括肠道细菌、真菌、病毒以及代谢产物,近期研究证实,人体微生态与多囊卵巢综合征的发生发展密切相关,肠道细菌可以转化胆汁酸影响宿主天然免疫系统,进而调节胰岛素抵抗,影响多囊卵巢综合征的发生发展。因此,胆汁酸在体内的丰度变化有望多囊卵巢综合征的表征或诊断提供新的路径。
发明内容
本发明提供一组可用于检测多囊卵巢综合征不同代谢亚型的生物标志物,进一步提供包含该等生物标志物的用于检测多囊卵巢综合征的试剂盒及其使用方法。
本发明的一个方面,提供了一组可用于多囊卵巢综合征诊断的生物标志物,所述生物标志物包括多种代谢物,所述的代谢物选自胆汁中的胆酸组分,其在至少一种靶血浆或血清中与在一种对照血浆或血清中差异表达。所述生物标志物可任选地与临床指标结合用于受试体多囊卵巢综合征。
在一种实施方案中,本发明生物标志物指受试体生物样本中存在的代谢物成分,受试体可以是人类或哺乳动物。生物样本可选自来源于受试体的血浆或血清。优选的,所述生物样本为血清。
在一种实施方案中,所述代谢物选自下列代谢物中一种或多种:胆酸、甘氨去氧胆酸、甘氨猪去氧胆酸、甘氨胆酸、甘氨熊去氧胆酸、牛磺猪去氧胆酸、牛磺胆酸、7-酮胆酸、牛磺熊去氧胆酸。优选的,所述代谢物为胆酸、甘氨去氧胆酸、牛磺熊去氧胆酸。
在一种实施方案中,所述的临床指标组合包含至少一个临床指标,其在至少一个多囊卵巢综合征患者中与在一个健康者中差异表达。临床指标可以按照常规临床检测方法获得,例如卵泡刺激素(FSH),黄体生成素(LH),黄体生成素/卵泡刺激素比值,性激素结合球蛋白(SHBG),脱氢表雄酮(DHEA),雌二醇(E2),睾酮(T), 雄烯二酮(AND),游离雄激素指数(FAI),空腹血糖,空腹胰岛素,窦卵泡数,多毛评分等。
在一种实施方案中,所述的生物标志物用于制备测试受试体多囊卵巢综合征的试剂盒的用途,所述试剂盒的使用方法包括,可通过测试受试体血浆或血清中代谢物的水平,任选地结合临床指标,测试受试体多囊卵巢综合征,所述临床指标优选受试体多毛评分,游离雄激素指数,空腹胰岛素。
本发明的另一方面,提供包含胆酸、甘氨去氧胆酸、牛磺熊去氧胆酸三种代谢物的可用于多囊卵巢综合征诊断试剂盒,所述试剂盒包含胆酸、甘氨去氧胆酸、牛磺熊去氧胆酸的标准溶液和内标标准溶液,所述内标标准溶液指同位素标记的胆酸、甘氨去氧胆酸、牛磺熊去氧胆酸,同位素标记方式可以是2H或13C。
在一种实施方案中,所述试剂盒还包含萃取液,萃取液由甲醇和乙腈组成,甲醇和乙腈的体积比为:1:1-5:1。进一步地,所述试剂盒还包含700μL的96孔板、350μL的V型96 孔板、封板硅胶、96孔密封铝膜。
本发明的另一方面,提供上述试剂盒用于诊断多囊卵巢综合征的使用方法,具体为,通过测定受试体的血清或血浆中代谢物胆酸、甘氨去氧胆酸、牛磺熊去氧胆酸的水平,并将这些测定值输入随机森林模型得到分值截断值进行判定。
在一种实施方案中,上述试剂盒用于诊断多囊卵巢综合征的使用方法,包括下列步骤: a)配制不同浓度的代谢产物标准品溶液:分别以胆酸、甘氨去氧胆酸、牛磺熊去氧胆酸的标准溶液配制不同浓度的溶液,与空白对照一起置于离心管,于台式离心机4000-10000转下离心10-30分钟;向各离心管加入新鲜制备的去离子水200微升,剧烈振荡,在800-1200转/分钟振荡溶解10-15分钟,静置,备用; b)内标标准品溶液配制:取3毫升的甲醇作内标稀释液,加入到内标准品溶液中,盖上盖子,剧烈振荡,静置约15分钟溶解。稀释内标溶液,内标溶液加到96孔微孔板; c)血清或血浆样本的制备:取出试剂盒提供的700微升微孔板,在A1到A8孔中依次加入5微升标准品1到标准品7和空白对照,在其他孔中加入5微升血清(或血浆)样品或5微升低、中、高浓度质控品。在各孔中加入25微升的内标准品溶液,盖上硅胶盖,1000rpm振荡10min。2000g离心2分钟。轻轻取下硅胶盖,避免微孔板中液体溅出,妥善放置硅胶垫防止污染,备用;30℃1450rpm反应60分钟,小心取下硅胶垫,妥善放置硅胶垫防止污染,备用;各孔加入350微升上样缓冲液,盖上硅胶盖剧烈振荡;-20℃放置20分钟,2000g离心20分钟;取下硅胶盖,小心吸取150微升上清至干净的V型底微孔板中,盖上铝箔封套,放入自动进样器中;d)将c)中制得的样本以液相色谱和质谱进行测定,计算样本中代谢产物的浓度; e)将d)中计算得到的生物标志物的浓度输入随机森林模型进行计算,按照分值对受试体的多囊卵巢综合征进行判定。
在一种实施方案中,本发明应用的随机森林模型可以是市场上可提供的软件,这些软件也可以任选地作为上述试剂盒的一部分。作为优选方案,软件包作为试剂盒的一部分。
附图说明
附图用来提供对本发明的进一步理解,并且构成说明书的一部分,与本发明的实施例一起用于解释本发明,并不构成对本发明的限制。在附图中:
图1A:临床指标的诊断价值的ROC曲线图;B:胆汁酸组合的诊断价值的ROC曲线图;C:临床+胆酸组合的诊断价值的ROC曲线图;
图2胆汁酸组合的诊断价值的ROC曲线图;
图3胆汁酸结合临床指标组合的诊断能力的ROC曲线图。
具体实施方式
以下结合附图对本发明的优选实施例进行说明,应当理解,此处所描述的优选实施例仅用于说明和解释本发明,并不用于限定本发明。
实施例中未注明具体条件的实验方法,通常按照常规条件,例如教科书和实验指南中所述的条件,或按照制造厂商所建议的条件。
实施例1:差异性生物标志物的筛选
本发明中的试验样本得到本地伦理委员会的批准并获得所有受试者的知情同意书。本发明共入组93名受试者,使用超高效液相色谱串联质谱技术分别检测了43例健康人和50例经证实的多囊卵巢综合征患者空腹(12小时)血清标本中胆酸的含量,以及相应临床指标的检测。检测方法如下所述。检测结果见表1。
1. 血清样本的采集和制备
采集空腹静脉血5mL置塑料离心管中。
血清制备:
1)缓慢颠转血清制备管5次。
2)在室温下(大约25摄氏度),将试管垂直置于试管架上1 .5小时。
3)将试管在2500rpm转速下离心10分钟(4摄氏度)。
4)将上清(约2 .5毫升)用移液器分装到塑料离心管(eppendorf,1 .5ml离心管)中,每只冻存管0 .5ml血清。
5)在离心管上标好样品号码。
6)迅速放入-80摄氏度冰箱。
2 .血清临床标志物检测
使用LH750血液学分析仪和Synchron DXC800临床系统根据制造商的试验方案进行血液学和生物化学检测。具体检测指标列于表1。
表1. 临床检测指标
序号 | PCOS | BMI | 年龄 | 腰臀比 | FSH | LH | LH/FSH | E2 | AND | T | SHBG | FAI | DHEA | 血糖 | 胰岛素 | HOMA | CHO | TG | HDL | LDL |
C001 | 2 | 35.7813 | 38 | 0.89167 | 4.54 | 3.34 | 0.74 | 49.57 | 2.04 | 0.38 | 25 | 5.24 | 2465.6 | 4.5 | 5.31 | 1.06 | 3.19 | 0.98 | 1.27 | 1.78 |
C005 | 2 | 21.2654 | 28 | 0.75789 | 8.34 | 3.64 | 0.44 | 29.42 | 1.01 | 0.29 | 35.3 | 2.86 | 1876.8 | 5.4 | 11.19 | 2.69 | 4.27 | 2.56 | 0.93 | 2.64 |
C006 | 2 | 23.3067 | 24 | 0.93333 | 3.24 | 4.73 | 1.46 | 27.78 | 3.06 | 0.38 | 84 | 1.56 | 2097.6 | 4.7 | 6.33 | 1.32 | 4.09 | 0.89 | 1.86 | 2 |
C007 | 2 | 26.782 | 35 | 0.81132 | 7.69 | 8.27 | 1.08 | 41.4 | 2.33 | 0.48 | 49.7 | 3.32 | 2281.6 | 4.5 | 10.3 | 2.06 | 4.34 | 0.55 | 1.23 | 3.49 |
C011 | 2 | 26.6389 | 31 | 0.78641 | 8.56 | 4.69 | 0.55 | 52.02 | 1.98 | 0.53 | 35.3 | 5.21 | 2428.8 | 4.7 | 4.77 | 1 | 5.51 | 0.5 | 1.56 | 3.71 |
C013 | 2 | 21.7993 | 27 | 0.82828 | 12.5 | 5.16 | 0.41 | 46.3 | 1.62 | 0.43 | 65.9 | 2.28 | 2760 | 4.5 | 4 | 0.8 | 5.08 | 0.55 | 1.95 | 2.71 |
C014 | 2 | 25.2826 | 30 | 0.88462 | 6.96 | 3.99 | 0.57 | 64.28 | 1.3 | 0.23 | 58.4 | 1.37 | 1840 | 4.7 | 5.04 | 1.05 | 5.42 | 0.58 | 1.82 | 3.38 |
C017 | 2 | 18.1429 | 28 | 0.85366 | 5.38 | 5.39 | 1 | 44.94 | 1.98 | 0.35 | 59.2 | 2.04 | 1987.2 | 5.4 | 8.97 | 2.15 | 4.51 | 0.93 | 1.29 | 2.84 |
C019 | 2 | 19.9481 | 27 | 0.80208 | 8.74 | 6.16 | 0.7 | 34.05 | 2.18 | 0.38 | 77.6 | 1.69 | 1656 | 4.6 | 3.37 | 0.69 | 4.88 | 0.92 | 2.04 | 2.62 |
C022 | 2 | 20.8333 | 34 | 0.88421 | 7.85 | 4.8 | 0.61 | 39.77 | 1.29 | 0.49 | 56.5 | 3.03 | 2870.4 | 5.2 | 7.5 | 1.73 | 4.43 | 0.61 | 1.68 | 2.67 |
C023 | 2 | 18.132 | 28 | 0.76404 | 6.34 | 2.82 | 0.44 | 31.87 | 2.98 | 0.24 | 58.2 | 1.42 | 1692.8 | 4.8 | 8.7 | 1.86 | 3.5 | 1.37 | 1.27 | 1.98 |
C024 | 2 | 20.9366 | 25 | 0.74747 | 4.89 | 2.61 | 0.53 | 70.55 | 1.2 | 0.29 | 58.8 | 1.73 | 1472 | 4 | 2.79 | 0.5 | 4.37 | 0.99 | 1.8 | 2.17 |
C026 | 2 | 27.3909 | 32 | 0.96078 | 5.98 | 4.75 | 0.79 | 39.22 | 1.36 | 0.46 | 78.8 | 2.04 | 3312 | 4.9 | 3.27 | 0.71 | 4.37 | 0.55 | 1.56 | 2.75 |
C031 | 2 | 18.5078 | 34 | 0.76923 | 6.06 | 5.35 | 0.88 | 65.92 | 1.54 | 0.5 | 89 | 1.96 | 1803.2 | 4.1 | 4.27 | 0.78 | 4.38 | 0.53 | 1.56 | 3.07 |
C032 | 2 | 21.4844 | 36 | 0.83 | 6.1 | 6.79 | 1.11 | 32.96 | 1.9 | 0.49 | 45.1 | 3.73 | 2392 | 4.1 | 4.97 | 0.91 | 3.25 | 0.45 | 1.5 | 1.73 |
C034 | 2 | 18.4911 | 28 | 0.88 | 8.91 | 6.89 | 0.77 | 35.95 | 2.4 | 0.38 | 53.5 | 2.45 | 3164.8 | 5.7 | 3.32 | 0.84 | 4.06 | 0.51 | 1.9 | 2 |
C037 | 2 | 21.4844 | 35 | 0.93333 | 8.65 | 1.62 | 0.19 | 57.74 | 2 | 0.46 | 63.7 | 2.5 | 3827.2 | 4.9 | 7.53 | 1.64 | 4.34 | 0.83 | 1.52 | 2.54 |
C039 | 2 | 18.3655 | 32 | 0.79545 | 3.23 | 2.05 | 0.63 | 67.01 | 1.12 | 0.48 | 45 | 3.69 | 2244.8 | 5.3 | 0.35 | 0.08 | 4.48 | 1.34 | 1.34 | 2.93 |
C042 | 2 | 24.4646 | 29 | 0.76042 | 5.61 | 4.08 | 0.73 | 46.85 | 2.38 | 0.65 | 39.7 | 5.72 | 1030.4 | 4.7 | 10.21 | 2.13 | 4.96 | 1.09 | 1.5 | 3.22 |
C044 | 2 | 21.3382 | 28 | 0.73737 | 8.01 | 3.46 | 0.43 | 47.94 | 2.04 | 0.44 | 51.5 | 2.93 | 2723.2 | 5 | 7.09 | 1.58 | 4.14 | 0.68 | 1.1 | 2.92 |
C046 | 2 | 20.6852 | 26 | 0.80851 | 5.32 | 4.7 | 0.88 | 27.51 | 3.15 | 0.4 | 48.8 | 2.87 | 2870.4 | 4.7 | 7.57 | 1.581289 | 4.52 | 0.88 | 1.49 | 2.79 |
C049 | 2 | 25.5102 | 33 | 0.88 | 7.57 | 6.39 | 0.84 | 30.51 | 1.66 | 0.33 | 37.3 | 3.11 | 1288 | 4.8 | 6.41 | 1.37 | 3.88 | 0.67 | 1.43 | 2.23 |
C052 | 2 | 21.83 | 32 | 0.80851 | 6.62 | 7.21 | 1.09 | 37.32 | 1.86 | 0.6 | 80.1 | 2.58 | 2281.6 | 4.2 | 3.64 | 0.68 | 4.55 | 1.26 | 1.4 | 2.88 |
C053 | 2 | 25.3906 | 27 | 0.82075 | 5.98 | 4.82 | 0.81 | 51.48 | 1.8 | 0.43 | 72.5 | 2.07 | 1692.8 | 4.5 | 3.87 | 0.77 | 3.58 | 0.57 | 1.32 | 2.2 |
C056 | 2 | 20.5693 | 30 | 0.80851 | 8.94 | 3.53 | 0.39 | 58.83 | 4.27 | 0.8 | 36.2 | 7.71 | 2723.2 | 4.4 | 5.08 | 0.99 | 3.6 | 0.55 | 1.37 | 2.07 |
C060 | 2 | 24.0346 | 24 | 0.84694 | 5.14 | 1.78 | 0.35 | 70 | 1.86 | 0.57 | 29.5 | 6.75 | 2833.6 | 5 | 8.42 | 1.87 | 4.69 | 1.08 | 1.64 | 2.92 |
C061 | 2 | 18.7783 | 26 | 0.84444 | 6.42 | 6.7 | 1.04 | 25.09 | 1.7 | 0.3 | 31.6 | 3.32 | 3164.8 | 4.7 | 4.52 | 0.94 | 3.77 | 0.85 | 1.61 | 2.07 |
C067 | 2 | 20.202 | 24 | 0.70707 | 9.18 | 4.36 | 0.47 | 39.77 | 2.08 | 0.48 | 55.1 | 3.03 | 2760 | 6.1 | 5.99 | 1.62 | 4.42 | 0.65 | 1.37 | 2.96 |
C072 | 2 | 21.9671 | 25 | 0.74737 | 5.3 | 8.9 | 1.68 | 311.06 | 2.71 | 0.57 | 18.5 | 10.65 | 3128 | 5 | 34.8 | 7.73 | 4.12 | 1.23 | 1.65 | 2.76 |
C074 | 2 | 25.3444 | 26 | 0.84906 | 4.92 | 7.14 | 1.45 | 64.83 | 2.37 | 0.53 | 35.9 | 5.1 | 2244.8 | 5.4 | 13.5 | 3.24 | 3.92 | 1.45 | 1.48 | 3.02 |
C078 | 2 | 28.3203 | 34 | 0.90909 | 6.4 | 5.78 | 0.9 | 27.24 | 1.94 | 0.4 | 31.9 | 4.36 | 2502.4 | 4.8 | 4.85 | 1.03 | 2.96 | 1.13 | 1.19 | 1.61 |
C079 | 2 | 24.9135 | 25 | 0.69159 | 6.73 | 5.71 | 0.85 | 54.48 | 1.96 | 0.74 | 72 | 3.58 | 2612.8 | 4.1 | 3.7 | 0.67 | 3.93 | 0.75 | 1.57 | 2.07 |
C080 | 2 | 27.3438 | 33 | 0.8381 | 5.39 | 4.84 | 0.9 | 65.1 | 4.35 | 0.69 | 19.2 | 12.4 | 1508.8 | 4 | 12.83 | 2.28 | 4.16 | 0.85 | 1.84 | 2.96 |
C089 | 2 | 29.0543 | 32 | 0.9619 | 4.67 | 2.93 | 0.63 | 41.4 | 5.47 | 0.66 | 22.3 | 10.27 | 2870.4 | 4.4 | 11.08 | 2.17 | 3.89 | 0.97 | 1.21 | 2.47 |
C090 | 2 | 28.457 | 33 | 0.88679 | 8.77 | 6.13 | 0.7 | 43.31 | 2.63 | 0.49 | 39.3 | 4.3 | 2539.2 | 5 | 9.67 | 2.15 | 3.95 | 1.13 | 0.99 | 2.73 |
C091 | 2 | 22.231 | 34 | 0.75789 | 7.33 | 2.01 | 0.27 | 38.13 | 1.66 | 0.45 | 19.8 | 7.93 | 1619.2 | 4.8 | 7.24 | 1.54 | 4.79 | 1.75 | 1.35 | 2.55 |
C092 | 2 | 22.1003 | 32 | 0.81633 | 7.45 | 7.24 | 0.97 | 44.67 | 1.26 | 0.27 | 23.9 | 3.89 | 1177.6 | 5.2 | 7.29 | 1.68 | 4.38 | 0.98 | 1.64 | 3.02 |
C095 | 2 | 27.3909 | 30 | 0.9798 | 8.75 | 2.19 | 0.25 | 39.77 | 0.76 | 0.21 | 29.1 | 2.54 | 920 | 5.7 | 7.1 | 1.8 | 3.46 | 0.82 | 1.49 | 2.59 |
C1 | 2 | 27.5312 | 28 | 0.93878 | 5.37 | 6.86 | 1.28 | 61.56 | 2.2 | 0.42 | 31.3 | 4.6 | 1876.8 | 4.4 | 30.27 | 5.92 | 5.24 | 1.83 | 1.14 | 3.73 |
C6 | 2 | 28.578 | 27 | 0.94444 | 4.2 | 4 | 0.95 | 83.62 | 1.26 | 0.44 | 51.4 | 2.96 | 1950.4 | 4.3 | 14.31 | 2.73 | 4.17 | 1.84 | 1.32 | 2.66 |
C7 | 2 | 25.2174 | 28 | 0.89583 | 8.58 | 28.3 | 3.3 | 60.2 | 2.29 | 0.42 | 29.5 | 4.88 | 2097.6 | 5 | 11.29 | 2.51 | 5.45 | 0.75 | 1.53 | 3.42 |
C8 | 2 | 26.4463 | 28 | 0.7963 | 7.74 | 15.3 | 1.98 | 53.39 | 3.81 | 0.49 | 35.1 | 4.84 | 1987.2 | 5.6 | 5.91 | 1.47 | 3.59 | 0.6 | 1.35 | 2.17 |
C9 | 2 | 28.959 | 26 | 0.84 | 7.03 | 14.7 | 2.09 | 56.66 | 3.15 | 0.37 | 47.6 | 2.67 | 1214.4 | 4.8 | 13.26 | 2.83 | 54 | 1.15 | 1.57 | 3.2 |
P001 | 1 | 26.5625 | 29 | 0.81553 | 6.08 | 4.56 | 0.75 | 60.47 | 2.38 | 0.58 | 20.1 | 10 | 3091.2 | 4.8 | 18.23 | 3.89 | 4.63 | 2.38 | 0.84 | 3.2 |
P002 | 1 | 23.4375 | 28 | 0.90722 | 7.35 | 3.35 | 0.46 | 34.05 | 0.9 | 0.31 | 22.5 | 4.76 | 1876.8 | 4.6 | 12.73 | 2.6 | 4.56 | 2.16 | 1.08 | 3.12 |
P003 | 1 | 22.8928 | 28 | 0.81053 | 3.56 | 2.23 | 0.63 | 49.57 | 3.04 | 0.82 | 8 | 35.63 | 3385.6 | 4.4 | 17.74 | 3.47 | 8.34 | 1.5 | 1.04 | 7.17 |
P005 | 1 | 19.2837 | 31 | 0.77907 | 5.93 | 3.46 | 0.58 | 24.9 | 1.3 | 0.66 | 28.5 | 8.04 | 2171.2 | 5.2 | 21.68 | 5.01 | 4.97 | 1.15 | 1.31 | 3.2 |
P006 | 1 | 22.3757 | 27 | 0.84694 | 7.74 | 11.1 | 1.43 | 59.92 | 4.12 | 0.82 | 19.6 | 14.54 | 3643.2 | 4.9 | 6.54 | 1.42 | 5.04 | 1.35 | 1.34 | 3.35 |
P007 | 1 | 25.9516 | 27 | 0.8381 | 5.85 | 14.5 | 2.48 | 108.95 | 3.58 | 0.42 | 17.1 | 8.42 | 3201.6 | 5.4 | 29.14 | 6.99 | 5.43 | 4.06 | 1.2 | 3.13 |
P008 | 1 | 24.8016 | 30 | 0.90625 | 5.01 | 8.76 | 1.75 | 62.38 | 4.21 | 0.76 | 16.4 | 16.1 | 3348.8 | 4.3 | 11.96 | 2.29 | 8.34 | 1.5 | 1.04 | 7.17 |
P009 | 1 | 31.4417 | 30 | 0.90385 | 4.96 | 6.05 | 1.22 | 46.85 | 2.45 | 0.49 | 12.7 | 13.39 | 2465.6 | 4.4 | 7.67 | 1.5 | 5.28 | 2.77 | 1.16 | 3.56 |
P010 | 1 | 23.3067 | 29 | 0.84444 | 5.39 | 9.88 | 1.83 | 49.57 | 2.28 | 0.81 | 25.5 | 10.98 | 3496 | 4.2 | 16.1 | 3.01 | 4.78 | 1.87 | 1.29 | 2.78 |
P011 | 1 | 21.875 | 28 | 0.87097 | 4.68 | 12.7 | 2.71 | 75.99 | 2.12 | 0.41 | 22.9 | 6.24 | 2760 | 5.2 | 9.17 | 2.12 | 5.3 | 1.13 | 1.46 | 3.76 |
P012 | 1 | 23.6614 | 33 | 0.88776 | 3.15 | 2.27 | 0.72 | 68.1 | 10.02 | 0.67 | 23.8 | 9.83 | 2723.2 | 4.6 | 9.8 | 2 | 2.97 | 1.34 | 1.06 | 1.38 |
P015 | 1 | 26.1719 | 30 | 0.91667 | 4.56 | 2.95 | 0.65 | 88.52 | 2.26 | 0.57 | 41.3 | 4.77 | 2134.4 | 5.1 | 8.78 | 1.99 | 8.85 | 1.93 | 1.19 | 6.86 |
P016 | 1 | 26.8386 | 35 | 0.86735 | 1.69 | 1.79 | 1.06 | 123.93 | 1.32 | 0.92 | 15.7 | 20.25 | 2760 | 4.4 | 8.55 | 1.67 | 5.92 | 2.95 | 1.46 | 4.11 |
P018 | 1 | 26.5338 | 28 | 0.83178 | 5.41 | 3.43 | 0.63 | 53.11 | 2.35 | 0.56 | 20.5 | 9.51 | 2392 | 5.6 | 14.8 | 3.68 | 5.32 | 1.77 | 0.94 | 3.77 |
P019 | 1 | 24.4141 | 32 | 0.85263 | 4.82 | 9.92 | 2.06 | 180.04 | 2.53 | 0.48 | 22 | 7.59 | 2539.2 | 5.2 | 18.04 | 4.17 | 4.86 | 1.02 | 1.37 | 3.37 |
P020 | 1 | 21.3593 | 36 | 0.8046 | 6.06 | 4.31 | 0.71 | 47.12 | 2.38 | 0.6 | 10.6 | 19.62 | 2686.4 | 5.2 | 11.78 | 2.72 | 5.62 | 3.59 | 0.99 | 3.59 |
P021 | 1 | 22.6003 | 31 | 0.86022 | 5.75 | 1.55 | 0.27 | 48.21 | 3.15 | 0.92 | 18.9 | 16.93 | 4305.6 | 5.2 | 11.09 | 2.56 | 4.88 | 1.14 | 1.2 | 3.35 |
P022 | 1 | 23.1247 | 32 | 0.82979 | 6.72 | 14.8 | 2.2 | 46.03 | 3.67 | 0.53 | 9.8 | 18.88 | 1876.8 | 4.9 | 10.26 | 2.23 | 3.62 | 3.38 | 0.86 | 2.09 |
P023 | 1 | 29.2421 | 33 | 0.88462 | 9.88 | 17.4 | 1.76 | 53.66 | 3.29 | 0.46 | 24.9 | 6.39 | 1987.2 | 5 | 10.76 | 2.39 | 3.33 | 5.37 | 0.71 | 1.13 |
P024 | 1 | 26.3672 | 32 | 0.83838 | 11.3 | 7.58 | 0.67 | 38.41 | 3.06 | 0.41 | 62.1 | 2.29 | 2576 | 5.3 | 10.87 | 2.56 | 5.07 | 0.66 | 1.67 | 3.19 |
P025 | 1 | 30.4577 | 27 | 0.86408 | 7.7 | 20.1 | 2.61 | 45.22 | 7.33 | 0.52 | 21.3 | 8.46 | 3312 | 4.8 | 3.62 | 0.77 | 5.6 | 1.13 | 1.3 | 3.8 |
P026 | 1 | 22.0317 | 29 | 0.86022 | 7.38 | 5.76 | 0.78 | 26.88 | 2.21 | 0.56 | 47.6 | 4.05 | 2944 | 4.4 | 7.36 | 1.44 | 5.13 | 0.89 | 2.37 | 1.97 |
P028 | 1 | 25.5937 | 34 | 0.97917 | 5.66 | 9.06 | 1.6 | 25.99 | 5.38 | 0.73 | 44.2 | 5.72 | 1288 | 5.3 | 5.14 | 1.21 | 3.98 | 1.29 | 1.46 | 2.27 |
P030 | 1 | 25.7778 | 38 | 0.88298 | 4.05 | 2.3 | 0.57 | 39.22 | 6.07 | 0.33 | 47.7 | 2.39 | 2944 | 5.2 | 15.8 | 3.65 | 6.05 | 1.39 | 1.41 | 4.13 |
P031 | 1 | 19.487 | 27 | 0.72043 | 5.3 | 3.5 | 0.66 | 8.17 | 1.48 | 0.58 | 21.4 | 9.36 | 3275.2 | 4.9 | 10.21 | 2.22 | 5.49 | 1.32 | 1.21 | 2.98 |
P032 | 1 | 25.0865 | 30 | 0.8932 | 4.7 | 7.3 | 1.55 | 46.85 | 3.09 | 0.67 | 22.6 | 10.21 | 2723.2 | 6 | 22.44 | 5.98 | 7.67 | 5.14 | 0.9 | 5.75 |
P035 | 1 | 21.5139 | 31 | 0.72043 | 3.96 | 9.46 | 2.39 | 61.56 | 6.44 | 0.51 | 20.6 | 8.54 | 3091.2 | 5.1 | 22.96 | 5.2 | 5.05 | 2.25 | 1.43 | 2.99 |
P036 | 1 | 20.1951 | 29 | 0.78889 | 5.3 | 13.2 | 2.49 | 96.42 | 2.71 | 0.76 | 71.3 | 3.69 | 2502.4 | 5.2 | 13.76 | 3.18 | 5.56 | 1.27 | 1.22 | 3.94 |
P039 | 1 | 29.7339 | 29 | 0.83495 | 5.17 | 6.52 | 1.26 | 50.66 | 2.14 | 0.42 | 20.1 | 7.21 | 6366.4 | 4.8 | 9.95 | 2.12 | 5.05 | 0.79 | 1.33 | 3.38 |
P041 | 1 | 26.0375 | 32 | 0.89899 | 6.18 | 14.3 | 2.31 | 46.58 | 3.01 | 0.67 | 33.4 | 6.92 | 2796.8 | 4.7 | 7.66 | 1.6 | 6.1 | 2.74 | 1.17 | 4.41 |
P042 | 1 | 28.959 | 31 | 0.93458 | 5.21 | 21.4 | 4.11 | 229.07 | 3.78 | 0.5 | 16.9 | 10.2 | 2944 | 4.5 | 6.5 | 1.3 | 4.15 | 0.55 | 1.68 | 2.72 |
P045 | 1 | 27.3438 | 26 | 0.94845 | 1.25 | 2.38 | 1.9 | 205.92 | 2.29 | 0.3 | 17.7 | 5.88 | 2281.6 | 5 | 24 | 5.33 | 5.03 | 1.45 | 1.06 | 4.24 |
P053 | 1 | 19.8347 | 27 | 0.77895 | 6.4 | 2.88 | 0.45 | 33.78 | 1.94 | 1.09 | 17.7 | 21.36 | 3974.4 | 4.7 | 12.24 | 2.56 | 4.3 | 1.54 | 1.17 | 2.93 |
P055 | 1 | 25.7657 | 35 | 0.9 | 7.94 | 10.6 | 1.34 | 40.31 | 3.21 | 0.59 | 26.4 | 7.69 | 1324.8 | 4.7 | 20.15 | 4.21 | 4.31 | 1.39 | 1.02 | 2.95 |
P057 | 1 | 20.6612 | 31 | 0.83333 | 7.25 | 5.58 | 0.77 | 56.38 | 1.26 | 0.25 | 83.4 | 1.03 | 1214.4 | 5.8 | 12.41 | 3.2 | 4.09 | 1.01 | 1.48 | 2.35 |
P058 | 1 | 27.1809 | 25 | 0.9604 | 7.42 | 7.67 | 1.03 | 88.59 | 5.58 | 0.54 | 25.8 | 7.21 | 3900.8 | 5.1 | 69.27 | 15.7 | 5.63 | 2.41 | 1.27 | 4.28 |
P059 | 1 | 26.8686 | 33 | 0.90099 | 6.15 | 19.8 | 3.22 | 82.8 | 3.24 | 0.78 | 18 | 15 | 4600 | 4.8 | 7.89 | 1.68 | 5.03 | 0.95 | 1.46 | 3.14 |
P062 | 1 | 30.4832 | 35 | 0.89189 | 5.88 | 2.7 | 0.46 | 56.66 | 1.88 | 0.82 | 19.9 | 14.21 | 4379.2 | 6.3 | 29.46 | 8.25 | 5.54 | 1.24 | 1.97 | 3.53 |
P065 | 1 | 22.8624 | 28 | 0.84656 | 7.03 | 3.92 | 0.56 | 31.32 | 2.45 | 0.61 | 17.3 | 12.14 | 3054.4 | 4.9 | 13.35 | 2.91 | 4.29 | 0.75 | 1.75 | 2.52 |
P066 | 1 | 18.4265 | 30 | 0.73333 | 7.14 | 15.2 | 2.13 | 66.73 | 5.1 | 1.73 | 46.3 | 12.98 | 3054.4 | 4.6 | 10.85 | 2.22 | 5.02 | 0.82 | 1.59 | 3.32 |
P073 | 1 | 27.6361 | 23 | 0.87745 | 4.7 | 4.63 | 0.99 | 46.03 | 1.58 | 0.78 | 16.7 | 16.2 | 6918.4 | 4 | 12.59 | 2.24 | 4.42 | 1.9 | 0.91 | 3.32 |
P076 | 1 | 20.4444 | 30 | 0.87356 | 5.39 | 9.69 | 1.8 | 79.81 | 3.55 | 0.39 | 39 | 3.49 | 2612.8 | 4.9 | 7.8 | 1.7 | 4.64 | 0.63 | 1.83 | 2.27 |
P078 | 1 | 34.7656 | 29 | 0.92857 | 5.49 | 13.8 | 2.51 | 40.86 | 1.62 | 0.6 | 25.4 | 8.2 | 2760 | 4.8 | 4.93 | 1.05 | 4.44 | 2.37 | 1.15 | 2.71 |
P080 | 1 | 25.9107 | 28 | 0.91429 | 3.66 | 13.85 | 3.78 | 63 | 4.27 | 0.66 | 73.3 | 3.1 | 1913.6 | 3.8 | 5.63 | 0.95 | 4.38 | 0.56 | 1.5 | 2.64 |
P082 | 1 | 22.4813 | 28 | 0.85106 | 7.33 | 4.36 | 0.59 | 40.58 | 5.41 | 0.59 | 49.7 | 4.1 | 2870.4 | 5.2 | 10.03 | 2.32 | 4.54 | 0.64 | 1.65 | 2.33 |
P083 | 1 | 27.0993 | 26 | 0.96078 | 4.54 | 3.34 | 0.74 | 49.57 | 2.04 | 0.38 | 25 | 5.24 | 2465.6 | 4.5 | 5.31 | 1.06 | 3.19 | 0.98 | 1.27 | 1.78 |
P084 | 1 | 21.9076 | 31 | 0.8125 | 8.34 | 3.64 | 0.44 | 29.42 | 1.01 | 0.29 | 35.3 | 2.86 | 1876.8 | 5.4 | 11.19 | 2.69 | 4.27 | 2.56 | 0.93 | 2.64 |
P086 | 1 | 27.3438 | 34 | 0.78704 | 3.24 | 4.73 | 1.46 | 27.78 | 3.06 | 0.38 | 84 | 1.56 | 2097.6 | 4.7 | 6.33 | 1.32 | 4.09 | 0.89 | 1.86 | 2 |
P087 | 1 | 19.1953 | 28 | 0.76923 | 7.69 | 8.27 | 1.08 | 41.4 | 2.33 | 0.48 | 49.7 | 3.32 | 2281.6 | 4.5 | 10.3 | 2.06 | 4.34 | 0.55 | 1.23 | 3.49 |
P093 | 1 | 20.7008 | 23 | 0.81395 | 8.56 | 4.69 | 0.55 | 52.02 | 1.98 | 0.53 | 35.3 | 5.21 | 2428.8 | 4.7 | 4.77 | 1 | 5.51 | 0.5 | 1.56 | 3.71 |
PCOS指标中,“1”代表确诊PCOS,“2”代表健康志愿者
3. 血清样本的胆酸检测:
样本制备:取100μl血清于1 .5mL的离心管中,加入150μL甲醇(含内标,50nM d4-CA (胆酸) ,d4-UDCA(熊去氧胆酸) ,d4-LCA(甘氨去氧胆酸))。漩涡震荡混匀10分钟,静置10分钟,然后4度13500转离心20分钟,取上清液进行UPLC-TQMS(超高效液相色谱-三重四级杆质谱) 分析。
分析仪器测试:UPLC-TQMS:采用沃特斯超高效液相色谱系统(沃特斯公司,美国),配备二元溶剂控制器和样本控制室。采用沃特斯XEVO三重四级杆质谱仪(沃特斯公司,美国),配备双电喷雾离子源。
色谱条件:采用UPLC BEH C18色谱柱(100mm×2 .1mm ,1 .7μm);柱温45℃;流动相A:水 (0 .1%甲酸),B:乙腈(0 .1%甲酸,);流速为0 .4mL/min;进样量为5uL;梯度洗脱条件:0-1min(5%B) ,1-5min(5-25%B) ,5-15 .5min(25-40%B) ,15 .5-17 .5min(40-95%B) , 17 .5-19min(95%B) ,19-19 .5min(95-5%B) ,19 .6-21min(5%B)。
质谱条件:电喷雾离子源采用负离子扫描模式(ESI-),具体条件如下:毛细管电压1.2kV ,锥孔电压55V ,萃取锥孔电压4V ,离子源温度150℃,脱溶剂气温度550℃,反向锥孔气流 50L/h ,脱溶剂气650L/h ,低质量区分辨率4 .7 ,高质量区分辨率15 ,多反应检测模式采集数据。
待测诊断标志物浓度的确定:以待测诊断标志物的标准品溶液的浓度与对应的待测诊断标志物以及与待测诊断标志物一样的稳定同位素内标面积比绘制标准曲线,采用同位素内标定量测定。同时通过样品添加同位素内标对样品检测过程进行质量控制。具体检测指标列于表2。
表2. 血清胆汁酸检测指标
序号 | PCOS | UDCA | HDCA | CDCA | DCA | CA | LCA | GUDCA | GCDCA | GDCA | GCA | GLCA | TUDCA | TCDCA | TDCA | TCA |
C001 | 2 | 86.03 | 18.25 | 90.79 | 230.53 | 125.92 | 1.71 | 78.01 | 1670.38 | 622.69 | 817.25 | 22.38 | 14.43 | 352.74 | 290.45 | 125.38 |
C005 | 2 | 348.52 | 20.96 | 653.43 | 762.55 | 941.23 | 2.33 | 582.71 | 1190.17 | 515.75 | 159.43 | 12.93 | 0.36 | 51.26 | 21.73 | 8.85 |
C006 | 2 | 280.47 | 54.19 | 550.78 | 971.86 | 16.93 | 3.35 | 488.26 | 5241.79 | 115.87 | 23.74 | 5.86 | 15.47 | 268.71 | 95.52 | 193.34 |
C007 | 2 | 39.12 | 22.58 | 172.57 | 696.72 | 121.71 | 20.03 | 64.05 | 391.41 | 21.42 | 231.92 | 10.27 | 0.28 | 42.08 | 36.91 | 13.06 |
C011 | 2 | 446.73 | 16.84 | 1304.63 | 190.46 | 371.97 | 1.83 | 540.24 | 772.03 | 167.54 | 138.27 | 8.06 | 17.92 | 79.32 | 143.06 | 8.48 |
C013 | 2 | 63.93 | 65.25 | 307.38 | 607.41 | 277.34 | 1.54 | 426.07 | 2456.37 | 165.07 | 437.66 | 42.09 | 7.28 | 127.25 | 53.14 | 13.41 |
C014 | 2 | 78.96 | 52.53 | 125.21 | 622.05 | 83.37 | 2.66 | 49.42 | 105.92 | 52.61 | 19.89 | 6.14 | 23.81 | 3.26 | 11.73 | 8.24 |
C017 | 2 | 173.17 | 58.52 | 429.86 | 76.83 | 103.45 | 22.32 | 580.43 | 1371.04 | 233.79 | 1740.62 | 8.38 | 9.75 | 106.17 | 14.95 | 5.53 |
C019 | 2 | 112.25 | 22.99 | 52.45 | 114.31 | 111.03 | 2.07 | 115.17 | 864.24 | 49.71 | 130.03 | 9.38 | 12.87 | 76.64 | 31.39 | 6.81 |
C022 | 2 | 80.58 | 21.96 | 109.47 | 143.17 | 71.19 | 0.86 | 787.59 | 2758.02 | 733.63 | 232.35 | 5.59 | 41.96 | 134.42 | 27.05 | 6.37 |
C023 | 2 | 50.38 | 63.85 | 57.98 | 402.73 | 108.69 | 23.37 | 49.58 | 659.94 | 642.64 | 142.09 | 14.71 | 20.78 | 39.54 | 38.73 | 13.31 |
C024 | 2 | 18.91 | 57.12 | 72.04 | 706.85 | 115.21 | 25.39 | 298.03 | 2327.06 | 49.22 | 158.39 | 93.34 | 10.82 | 101.81 | 79.97 | 12.62 |
C026 | 2 | 90.12 | 40.47 | 122.73 | 541.98 | 239.14 | 8.05 | 524.06 | 385.65 | 275.93 | 65.97 | 25.41 | 15.88 | 14.02 | 5.89 | 2.43 |
C031 | 2 | 82.72 | 68.31 | 804.48 | 122.35 | 385.59 | 94.82 | 120.54 | 1226.87 | 157.91 | 173.42 | 114.65 | 43.76 | 37.43 | 17.07 | 6.39 |
C032 | 2 | 98.03 | 18.44 | 646.08 | 596.57 | 1103.27 | 28.81 | 483.62 | 345.54 | 237.67 | 78.04 | 17.89 | 17.62 | 15.73 | 8.74 | 2.09 |
C034 | 2 | 162.9 | 32.53 | 984.55 | 1300.32 | 18.37 | 26.08 | 250.96 | 745.69 | 128.38 | 194.71 | 18.61 | 50.19 | 30.74 | 40.26 | 5.83 |
C037 | 2 | 41.51 | 26.14 | 160.43 | 248.27 | 609.61 | 7.47 | 151.04 | 590.16 | 11.02 | 503.08 | 4.72 | 5.11 | 95.51 | 49.53 | 41.71 |
C039 | 2 | 85.68 | 42.65 | 151.96 | 312.48 | 129.89 | 17.53 | 135.88 | 340.12 | 474.59 | 38 | 29.47 | 45.97 | 11.71 | 16.39 | 15.45 |
C042 | 2 | 79.25 | 68.72 | 136.13 | 120.23 | 138.92 | 26.31 | 30.92 | 103.94 | 1510.96 | 1579.37 | 13.22 | 11.32 | 4.95 | 6.13 | 4.28 |
C044 | 2 | 5.31 | 19.41 | 56.14 | 244.34 | 96.53 | 25.95 | 16.19 | 138.81 | 148.22 | 43.28 | 1.35 | 16.28 | 17.96 | 36.32 | 17.37 |
C046 | 2 | 81.61 | 44.26 | 181.52 | 281.58 | 311.81 | 14.09 | 302.52 | 1872.65 | 2124.64 | 313.36 | 40.14 | 9.62 | 216.05 | 199.91 | 19.69 |
C049 | 2 | 2.97 | 9.35 | 3.94 | 587.12 | 18.04 | 1.53 | 18.83 | 190.84 | 161.75 | 20.52 | 7.56 | 16.33 | 20.18 | 39.64 | 18.74 |
C052 | 2 | 56.94 | 12.73 | 271.75 | 914.27 | 8.77 | 1.88 | 120.46 | 528.73 | 150.28 | 132.08 | 5.85 | 0.74 | 33.21 | 26.65 | 18.23 |
C053 | 2 | 3.77 | 1.88 | 33.18 | 63.23 | 978.58 | 0.69 | 36.38 | 199.42 | 137.61 | 50.71 | 0.69 | 11.19 | 11.27 | 24.98 | 18.22 |
C056 | 2 | 94.61 | 21.43 | 78.38 | 797.3 | 139.25 | 1.19 | 60.02 | 83.14 | 64.32 | 24.37 | 11.85 | 0.14 | 7.83 | 6.45 | 2.49 |
C060 | 2 | 35.75 | 40.39 | 93.02 | 170.72 | 197.28 | 10.06 | 46.39 | 213.28 | 921.59 | 84.21 | 6.14 | 10.13 | 12.15 | 13.89 | 3.21 |
C061 | 2 | 49.14 | 54.45 | 213.29 | 661.52 | 239.86 | 32.83 | 140.35 | 1185.86 | 104.35 | 166.75 | 8.04 | 7.32 | 227.53 | 215.62 | 20.12 |
C067 | 2 | 204.63 | 25.76 | 1787.72 | 20.92 | 1426.77 | 5.94 | 124.38 | 432.06 | 1512.43 | 80.51 | 10.02 | 12.04 | 30.34 | 48.26 | 4.67 |
C072 | 2 | 35.25 | 19.96 | 71.85 | 279.22 | 19.71 | 11.57 | 872.25 | 25.32 | 1250.89 | 21.84 | 1.19 | 0.69 | 2.83 | 16.51 | 12.93 |
C074 | 2 | 19.34 | 31.44 | 29.72 | 98.49 | 98.32 | 14.02 | 13.24 | 15.93 | 32.86 | 17.25 | 0.5 | 12.27 | 7.17 | 22.36 | 17.09 |
C078 | 2 | 72.49 | 22.99 | 209.18 | 346.52 | 130.51 | 13.89 | 307.43 | 970.02 | 887.15 | 133.49 | 4.75 | 23.97 | 41.76 | 36.15 | 4.19 |
C079 | 2 | 31.81 | 13.14 | 164.93 | 242.01 | 107.35 | 21.72 | 244.97 | 264.87 | 330.96 | 30.82 | 0.21 | 10.09 | 12.21 | 25.81 | 15.95 |
C080 | 2 | 5.08 | 2.35 | 5.19 | 31.67 | 32.95 | 1.96 | 39.43 | 72.92 | 111.83 | 28.27 | 0.41 | 13.59 | 10.89 | 36.41 | 16.48 |
C089 | 2 | 24.01 | 4.08 | 110.28 | 46.69 | 2467.04 | 5.74 | 106.51 | 575.59 | 196.78 | 81.19 | 1.34 | 20.07 | 78.27 | 53.83 | 21.77 |
C090 | 2 | 18.34 | 0.62 | 271.16 | 161.96 | 607.36 | 1.92 | 319.84 | 164.23 | 260.16 | 39.23 | 0.91 | 18.92 | 48.36 | 34.02 | 19.88 |
C091 | 2 | 29.39 | 34.73 | 159.45 | 453.07 | 107.75 | 10.75 | 102.92 | 938.37 | 698.04 | 116.53 | 5.46 | 47.38 | 177.83 | 190.97 | 37.07 |
C092 | 2 | 0.85 | 2.38 | 11.29 | 31.19 | 45.31 | 4.26 | 50.83 | 91.56 | 774.59 | 34.45 | 0.27 | 13.57 | 14.17 | 39.85 | 19.15 |
C095 | 2 | 43.01 | 3.14 | 29.13 | 701.29 | 29.56 | 3.12 | 27.25 | 40.57 | 33.07 | 9.48 | 0.79 | 43.28 | 7.99 | 18.37 | 15.92 |
C1 | 2 | 12.27 | 2.65 | 86.29 | 70.42 | 22.71 | 3.84 | 199.41 | 1168.56 | 944.05 | 252.58 | 2.67 | 19.45 | 83.07 | 74.32 | 28.06 |
C6 | 2 | 142.41 | 3.18 | 528.85 | 883.92 | 974.65 | 1.47 | 58.62 | 198.52 | 366.94 | 70.32 | 0.18 | 11.95 | 13.58 | 22.59 | 15.03 |
C7 | 2 | 4.74 | 0.91 | 215.68 | 94.51 | 59.37 | 3.09 | 952.76 | 102.17 | 310.49 | 292.57 | 0.11 | 18.81 | 8.64 | 19.26 | 16.18 |
C8 | 2 | 77.84 | 13.51 | 27.16 | 170.08 | 15.81 | 71.01 | 21.84 | 15.77 | 294.98 | 10.22 | 2.66 | 0.97 | 1.17 | 16.01 | 15.96 |
C9 | 2 | 68.53 | 12.98 | 158.67 | 263.36 | 166.52 | 38.98 | 228.07 | 1144.72 | 685.76 | 328.45 | 3.37 | 29.13 | 155.28 | 155.96 | 44.54 |
P001 | 1 | 99.49 | 102.46 | 414.95 | 1447.01 | 265.16 | 17.28 | 359.29 | 1575.06 | 98.36 | 323.68 | 15.35 | 0.58 | 78.96 | 44.17 | 13.89 |
P002 | 1 | 64.68 | 27.52 | 41.69 | 285.52 | 75.99 | 11.05 | 31.14 | 205.29 | 25.08 | 73.76 | 2.87 | 1.06 | 49.34 | 35.29 | 10.87 |
P003 | 1 | 74.42 | 69.01 | 813.53 | 104.35 | 138.93 | 84.72 | 238.11 | 1662.83 | 10.68 | 487.85 | 50.75 | 0.74 | 130.33 | 164.26 | 23.22 |
P005 | 1 | 108.02 | 33.08 | 917.59 | 641.07 | 443.86 | 51.79 | 417.24 | 1434.37 | 81.96 | 419.82 | 23.55 | 2.55 | 72.48 | 17.21 | 17.34 |
P006 | 1 | 25.36 | 38.87 | 14.64 | 107.14 | 182.15 | 23.49 | 54.32 | 440.31 | 475.96 | 135.64 | 10.09 | 3.44 | 20.54 | 8.27 | 7.02 |
P007 | 1 | 145.47 | 46.65 | 363.38 | 317.67 | 226.32 | 2.78 | 251.45 | 283.49 | 5.59 | 45.06 | 6.95 | 0.26 | 6.89 | 54.44 | 9.28 |
P008 | 1 | 22.49 | 52.24 | 110.93 | 450.57 | 116.78 | 14.57 | 22.85 | 1115.87 | 14.64 | 77.14 | 3.02 | 6.14 | 8.55 | 5.36 | 48.16 |
P009 | 1 | 29.41 | 30.91 | 862.86 | 857.53 | 379.22 | 11.31 | 292.19 | 2181.58 | 36.02 | 245.92 | 13.77 | 1.68 | 222.79 | 17.14 | 57.09 |
P010 | 1 | 136.84 | 15.03 | 407.01 | 123.89 | 133.95 | 11.49 | 241.78 | 250.38 | 73.06 | 57.12 | 9.32 | 1.18 | 24.83 | 92.39 | 4.45 |
P011 | 1 | 120.17 | 24.78 | 273.67 | 342.18 | 170.52 | 5.22 | 15.98 | 1923.34 | 8.06 | 858.91 | 2.53 | 11.06 | 120.02 | 64.53 | 28.02 |
P012 | 1 | 187.53 | 38.91 | 452.96 | 72.69 | 873.04 | 16.68 | 1064.91 | 1639.25 | 11.55 | 519.49 | 10.42 | 10.71 | 131.58 | 4.66 | 18.15 |
P015 | 1 | 61.28 | 67.03 | 198.62 | 816.02 | 145.81 | 20.92 | 287.12 | 518.72 | 9.08 | 284.62 | 4.74 | 40.36 | 29.11 | 19.31 | 29.02 |
P016 | 1 | 114.36 | 14.21 | 393.53 | 126.39 | 284.67 | 13.75 | 51.07 | 1361.06 | 10.34 | 620.07 | 11.02 | 7.02 | 141.46 | 13.75 | 34.52 |
P018 | 1 | 127.32 | 34.95 | 8.35 | 931.89 | 1001.79 | 17.34 | 440.11 | 914.04 | 83.87 | 210.69 | 22.49 | 0.48 | 145.23 | 72.91 | 23.13 |
P019 | 1 | 39.73 | 42.07 | 130.47 | 394.93 | 270.61 | 12.72 | 197.58 | 814.27 | 79.65 | 261.87 | 9.27 | 3.65 | 126.45 | 64.94 | 27.06 |
P020 | 1 | 45.01 | 48.25 | 204.16 | 772.61 | 345.02 | 19.68 | 407.13 | 1505.31 | 37.18 | 480.31 | 11.46 | 0.74 | 145.06 | 83.12 | 25.85 |
P021 | 1 | 57.61 | 30.43 | 354.31 | 524.51 | 226.93 | 4.23 | 266.95 | 549.39 | 36.52 | 216.02 | 5.15 | 4.28 | 87.28 | 39.74 | 25.59 |
P022 | 1 | 199.76 | 32.84 | 39.42 | 344.26 | 102.14 | 2.48 | 31.72 | 102.84 | 15.41 | 114.64 | 6.65 | 10.86 | 12.43 | 15.03 | 11.04 |
P023 | 1 | 23.62 | 27.57 | 62.27 | 509.16 | 124.32 | 17.36 | 133.05 | 309.87 | 56.58 | 225.77 | 7.27 | 0.74 | 44.57 | 51.67 | 20.95 |
P024 | 1 | 27.38 | 5.05 | 136.15 | 763.53 | 40.08 | 2.63 | 61.74 | 100.45 | 12.82 | 42.89 | 0.86 | 5.13 | 14.17 | 13.62 | 2.93 |
P025 | 1 | 219.37 | 51.13 | 705.48 | 640.31 | 201.58 | 20.75 | 544.95 | 778.69 | 51.67 | 272.68 | 4.33 | 5.29 | 124.82 | 29.98 | 22.58 |
P026 | 1 | 19.32 | 76.23 | 348.41 | 541.23 | 174.63 | 71.65 | 54.06 | 503.52 | 81.75 | 232.57 | 44.56 | 0.17 | 33.29 | 54.18 | 12.86 |
P028 | 1 | 558.78 | 17.26 | 903.09 | 504.06 | 144.07 | 19.18 | 481.13 | 304.68 | 233.55 | 60.51 | 4.97 | 2.78 | 19.12 | 12.25 | 9.64 |
P030 | 1 | 36.07 | 30.13 | 210.25 | 120.35 | 177.69 | 11.42 | 72.73 | 297.53 | 40.91 | 177.13 | 4.12 | 0.59 | 21.01 | 19.01 | 5.72 |
P031 | 1 | 6.38 | 15.34 | 59.64 | 193.73 | 22.43 | 3.02 | 31.94 | 169.47 | 13.59 | 17.32 | 3.03 | 4.28 | 11.35 | 14.97 | 35.28 |
P032 | 1 | 19.52 | 67.08 | 126.97 | 125.95 | 190.41 | 5.92 | 80.09 | 634.82 | 138.51 | 264.59 | 6.65 | 2.54 | 25.61 | 3.26 | 7.96 |
P035 | 1 | 74.34 | 18.97 | 220.74 | 208.76 | 164.35 | 14.73 | 65.35 | 130.83 | 562.41 | 36.45 | 7.18 | 10.33 | 2.87 | 30.24 | 4.89 |
P036 | 1 | 37.17 | 15.71 | 306.49 | 381.98 | 321.27 | 11.93 | 317.85 | 1077.49 | 65.1 | 299.92 | 11.85 | 8.12 | 71.11 | 20.58 | 13.43 |
P039 | 1 | 21.33 | 24.65 | 132.15 | 413.59 | 140.79 | 14.35 | 57.46 | 225.81 | 47.45 | 85.56 | 20.25 | 1.57 | 13.81 | 15.67 | 16.47 |
P041 | 1 | 13.15 | 94.78 | 849.63 | 780.34 | 183.32 | 1.37 | 15.02 | 357.46 | 31.17 | 58.04 | 8.29 | 5.09 | 6.44 | 56.16 | 22.59 |
P042 | 1 | 162.55 | 47.22 | 25.43 | 1639.13 | 1508.92 | 11.52 | 250.95 | 894.76 | 89.77 | 409.57 | 16.24 | 2.62 | 90.43 | 55.27 | 5.23 |
P045 | 1 | 43.46 | 20.69 | 344.59 | 603.92 | 227.35 | 8.94 | 27.74 | 1359.05 | 23.37 | 328.12 | 17.45 | 6.99 | 167.61 | 76.35 | 23.27 |
P053 | 1 | 117.92 | 59.97 | 99.06 | 441.79 | 1475.09 | 2.68 | 149.18 | 320.89 | 11.78 | 230.76 | 3.91 | 2.16 | 74.73 | 9.82 | 23.63 |
P055 | 1 | 22.45 | 0.71 | 184.51 | 283.84 | 105.68 | 6.92 | 122.67 | 350.24 | 34.28 | 104.92 | 2.68 | 1.22 | 26.49 | 13.76 | 6.81 |
P057 | 1 | 68.17 | 5.84 | 270.43 | 56.02 | 169.13 | 6.02 | 169.63 | 338.92 | 61.79 | 115.24 | 21.37 | 0.15 | 14.35 | 43.37 | 19.08 |
P058 | 1 | 18.96 | 21.78 | 135.72 | 363.26 | 107.26 | 3.56 | 66.81 | 347.24 | 36.25 | 130.37 | 2.69 | 0.81 | 21.93 | 10.37 | 5.81 |
P059 | 1 | 79.58 | 23.38 | 121.19 | 386.04 | 141.95 | 12.43 | 106.05 | 2099.64 | 10.41 | 64.23 | 2.91 | 1.18 | 7.28 | 123.43 | 0.82 |
P062 | 1 | 286.57 | 51.42 | 10.75 | 147.66 | 936.35 | 4.39 | 579.56 | 1353.21 | 24.75 | 643.59 | 4.08 | 34.74 | 129.17 | 40.67 | 28.58 |
P065 | 1 | 92.84 | 12.53 | 403.72 | 335.12 | 185.21 | 27.37 | 281.17 | 1022.29 | 18.81 | 275.14 | 7.63 | 4.29 | 56.81 | 90.98 | 13.67 |
P066 | 1 | 41.98 | 4.18 | 327.08 | 402.64 | 140.85 | 8.24 | 106.89 | 458.39 | 24.18 | 305.45 | 14.59 | 0.17 | 41.47 | 6.82 | 17.08 |
P073 | 1 | 86.89 | 27.33 | 341.96 | 1602.17 | 96.18 | 9.17 | 42.72 | 2157.35 | 11.67 | 28.45 | 2.68 | 45.91 | 7.48 | 26.59 | 18.11 |
P076 | 1 | 87.11 | 31.76 | 360.91 | 265.11 | 183.26 | 18.05 | 125.57 | 107.49 | 6.44 | 27.94 | 29.02 | 5.97 | 5.86 | 26.88 | 28.41 |
P078 | 1 | 231.07 | 3.06 | 150.79 | 140.75 | 148.04 | 2.73 | 113.74 | 377.91 | 42.35 | 127.25 | 19.67 | 7.73 | 52.72 | 19.49 | 11.68 |
P080 | 1 | 164.79 | 24.68 | 23.07 | 1074.79 | 696.03 | 11.64 | 19.32 | 480.59 | 55.49 | 141.94 | 44.73 | 3.15 | 42.87 | 23.52 | 11.42 |
P082 | 1 | 106.23 | 55.85 | 189.59 | 264.08 | 281.87 | 18.37 | 29.69 | 162.83 | 15.21 | 91.78 | 6.08 | 0.75 | 7.85 | 21.65 | 3.19 |
P083 | 1 | 386.14 | 15.09 | 292.87 | 163.75 | 1761.26 | 2.53 | 49.64 | 2859.38 | 155.26 | 828.01 | 4.64 | 7.94 | 261.36 | 54.96 | 37.16 |
P084 | 1 | 49.37 | 0.45 | 240.76 | 60.19 | 276.29 | 12.98 | 182.21 | 246.47 | 259.14 | 90.53 | 44.89 | 4.35 | 17.23 | 25.12 | 0.73 |
P086 | 1 | 234.73 | 42.85 | 295.43 | 433.21 | 1535.71 | 3.95 | 36.29 | 2636.35 | 13.16 | 1932.35 | 6.43 | 2.37 | 62.64 | 16.05 | 12.54 |
P087 | 1 | 14.35 | 7.84 | 42.51 | 1388.97 | 160.95 | 43.16 | 102.45 | 692.21 | 15.46 | 161.08 | 10.07 | 0.56 | 43.12 | 22.87 | 18.36 |
P093 | 1 | 45.52 | 22.31 | 218.62 | 1543.31 | 152.72 | 20.42 | 127.92 | 452.76 | 47.51 | 186.49 | 4.96 | 0.74 | 30.53 | 19.42 | 8.33 |
实施例2 胆酸组合及临床指标组合区分健康组和多囊卵巢综合征组
为了区分健康人和多囊卵巢综合征患者,我们使用单因素Wilcoxon秩和检验和LASSO来挑选和鉴定候选的生物标记物,并用随机森林模型来评价候选变量并建立模型。
随机森林模型建立方法及相关参数选择:相关模型建立在R语言的 R-Studio 环境下进行。首先对原始数据进行清洗预处理,本数据集的缺失数据采用的是随机森林迭代法的missForest( )方法填补缺失值。并且按照50/50的比例划分数据,建立训练集和测试集。进一步,我们使用e1071包构建SVM模型,实验中使用tune.svm函数选择调优参数,使用十折交叉验证对参数进行调优。最后,选择Sigmoid核函数作为最优的建模模型。
随机森林模型标记方法包括如下步骤:
1)获取健康人和多囊卵巢综合征数据,并对所获取到的样本数据进行归一化处理,且进一步将归一化处理后的样本数据划分出测试集和多个训练集;
2)选取决策树类型为CART分别对每一个训练集进行决策树训练,得到每一个训练集各自训练出的相应CART决策树模型,分别对每一个训练集进行决策树训练,得到每一个训练集各自训练出的相应CART决策树模型,即得到N-1个CART决策树模型。
3)通过特征重要性,对每一个CART决策树模型中所有特征均进行评估,得到每一个CART决策树模型中满足预定条件的特征集;具体过程为,首先,确定当前待评估的CART决策树模型,并在当前待评估的CART决策树模型中,汇总出对应训练集中训练数据的所有特征;其次,计算出当前待评估的CART决策树模型中每个特征的基尼指数评分,并待按照预设的特征重要性,对当前待评估的CART决策树模型中所有计算出的基尼指数评分进行排列后,得到当前待评估的CART决策树模型中按照特征重要性降序排列的特征集;最后,依据预设的剔除比例,重复在特征集中以特征重要性从小到大的顺序进行特征剔除,直至特征集中特征数量达到阈值(如m)为止,得到当前待评估的CART决策树模型中特征剔除后的特征集。其中,m可以设置为当前待评估的CART决策树模型中总特征数开平方。
通过上述随机森林模型,我们发现多毛评分、游离雄激素指数、空腹胰岛素等临床指标及胆酸汁对预测多囊卵巢综合征具有预测能力。具体如表3及图1所示。其中,表3中所述“所有胆汁酸”为人体所含内源性胆酸成分,包括熊去氧胆酸、猪去氧胆酸、鹅去氧胆酸、去氧胆酸、胆酸、石胆酸、甘氨熊去氧胆酸、甘氨鹅去氧胆酸、甘氨胆酸、牛磺熊去氧胆酸、牛磺鹅去氧胆酸、牛磺去氧胆酸、牛磺胆酸。
实施例3 特定胆酸组合区分健康组和多囊卵巢综合征组
样本及数据来源同实施例1。
在所有胆酸汁组分中,我们发现胆酸、甘氨脱氧胆酸、牛磺熊脱氧胆酸三种组分对预测多囊卵巢综合征起到了最为重要的作用。为了进一步验证三种组分的预测功能,我们继续用随机森林模型来评价候选变量并建立模型,模型建立方法同实施例2,验证胆酸、甘氨脱氧胆酸、牛磺熊脱氧胆酸组合对预测多囊卵巢综合征的预测能力,结果见表4。从表4可知,通过与现有的测试指标比较,这三种代谢物的组合具有更佳的预测能力。
如实施例1所述,50例多囊卵巢综合征患者患者和43例健康人,利用在生物标记物组合随机森林模型,可输出上述受试者患多囊卵巢综合征的可能性,并通过ROC分析中的约登最优点找到最佳截断值,可评估该模型对于区分患者和健康人的总能力。结果如图2所示: ROC曲线下面积和95%置信区间为0.932,最佳截断值为 0.513,最佳截断值处的灵敏度和特异性百分比分别为90 .9%和92.9%。如所述对象测定标志物水平,将这些测定值带入随机森林模型后,如果个体得分阈值小于0.513,表明此个体具有较高的患多囊卵巢综合征风险;如果个体得分阈值大于0.513,表明此个体具有较低的患多囊卵巢综合征风险。
实施例4 胆酸组合结合临床指标区分健康组和多囊卵巢综合征组
样本及数据来源同实施例1。
用随机森林模型来评价候选变量并建立模型,模型建立方法同实施例2,验证胆酸、甘氨脱氧胆酸、牛磺熊脱氧胆酸组合以及多毛评分,游离雄激素指数,空腹胰岛素对预测多囊卵巢综合征的预测能力,结果见表5。从表5可知,通过与现有的测试指标比较,这三种代谢物及临床指标的组合具有更佳的预测能力。
如实施例1所述, 50例多囊卵巢综合征患者患者和43例健康人,利用在生物标记物组合随机森林模型,可输出上述受试者患多囊卵巢综合征的可能性,并通过ROC分析中的约登最优点找到最佳截断值,可评估该模型对于区分患者和健康人的总能力。结果(图3):ROC曲线下面积和95%置信区间为0.9773(0.9471-1),最佳截断值为 0 .592,最佳截断值处的灵敏度和特异性百分比分别为92 .9%和90.9%。如所述对象测定标志物水平,将这些测定值带入随机森林模型后,如果个体得分阈值小于0 .592,表明此个体具有较高的患多囊卵巢综合征风险;如果个体得分阈值大于0 .592,表明此个体具有较低的患多囊卵巢综合征风险。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
Claims (6)
1.一种用于多囊卵巢综合征诊断的试剂盒,其特征在于,包含生物标志物的标准溶液和内标标准溶液,所述生物标志物包括胆酸、甘氨去氧胆酸和牛磺熊去氧胆酸,并且至少一种生物标志物在受试体生物样本中与对照样本中差异表达;所述受试体生物样本选自血浆或血清;所述内标标准溶液为经同位素标记的所述生物标志物溶液,同位素标记方式为2H或13C标记;所述生物标志物任选地与临床指标结合用于诊断受试体多囊卵巢综合征亚型。
2.如权利要求1所述的试剂盒,其特征在于,所述生物标志物进一步包括下列代谢物中一种或多种:熊去氧胆酸、猪去氧胆酸、鹅去氧胆酸、去氧胆酸、石胆酸、甘氨熊去氧胆酸、甘氨鹅去氧胆酸、甘氨胆酸、牛磺鹅去氧胆酸、牛磺去氧胆酸和牛磺胆酸,所述临床指标选自受试者血浆如下指标中的一种或多种:卵泡刺激素,黄体生成素,黄体生成素/卵泡刺激素比值,性激素结合球蛋白,脱氢表雄酮,雌二醇,睾酮, 雄烯二酮,游离雄激素指数,空腹血糖,空腹胰岛素,窦卵泡数,以及多毛评分。
3.如权利要求1或2所述的试剂盒,其特征在于,所述试剂盒还包含萃取液,萃取液由甲醇和乙腈组成,甲醇和乙腈的体积比为:1:1-5:1。
4.如权利要求1所述的试剂盒的使用方法,其特征在于,通过测定受试体的血清或血浆中代谢物胆酸、甘氨脱氧胆酸、牛磺脱氧胆酸的摩尔浓度,并将这些测定值输入随机森林模型得到截断值进行判定。
5.如权利要求2所述的试剂盒的使用方法,其特征在于,通过测试受试体血浆或血清中生物标志物的水平,结合临床指标数值,将上述测试结果和临床指标数值输入随机森林模型进行统计计算。
6.如权利要求4或5所述的试剂盒的使用方法,包括下列步骤:
a)配制不同浓度的生物标志物标准品溶液;
b)配制内标标准品溶液配制;
c)制备受试体血清或血浆样本;
d)将c)中制得的样本以液相色谱和质谱进行测定,计算样本中代谢产物的浓度;
e)将d)中计算得到的生物标志物的浓度输入随机森林模型或梯度提升树模型进行计算,按照分值对受试体是否患有多囊卵巢综合征进行判定。
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