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Circulation(IF=23.054):更年期后的女性冠心病患者代谢组学研究

分类:行业资讯   发布时间 2020-05-18   阅读: 41

冠心病(CHD,coronary heart disease)目前仍然是导致全球女性和男性死亡的主要原因。然而冠心病在不同性别中的临床表现不同,女性表现出更高比例的非阻塞性动脉粥样硬化和微血管疾病以及死亡率[1]。糖尿病是女性冠心病患者的高风险因子,2型糖尿病女性比男性有大于40%概率的高风险易患冠心病[2,3]。此外,现在已有报道TCA循环代谢物、肠道菌群代谢物TMAO、脂类代谢物与冠心病事件发生密切相关,glutamate和glutamine以及芳香族氨基酸与糖尿病和冠心病密切相关[4-13]。但是,尽管已有证据表明性别会影响人体代谢物轮廓谱[14,15],而国际上使用代谢组学研究代谢物与女性患冠心病风险,尤其是女性绝经与冠心病之间的相关研究更是稀缺[16]。因此,本研究先采用LC-MS代谢组学技术去挖掘WHI (Women’s Health Initiative) [17]和 WHI-OS (Women’s Health Initiative-Observational Study) [18]人群队列中绝经女性冠心病事件与代谢物间的关系,然后在WHI-HT (Women’s Health Initiative-Hormone Trials)[19]人群队列中进行验证,最后在另一个独立的PREDIMED饮食干预 (Prevención con Dieta Mediterránea) [20,21]人群队列中评估了这些显著性关联物质在男女性别中的普适性。

本研究[22]从WHI-OS人群队列(含>93676位绝经女性)中挑选出472对绝经女性冠心病患者和健康对照绝经女性,匹配了种族、民族、子宫切除手术、年龄(±5年)、以及纳入队列时间(±2年)等因素;从WHI-HT人群队列(含>16608位绝经女性,>10739位子宫切除女性)中挑选出312位绝经女性冠心病和315位健康对照女性,匹配了种族、民族、子宫切除手术、年龄(±5年)以及纳入队列时间(±2年)等因素(如 table1 所示),从PREDIMED人群队列(含1017位男性和女性地中海饮食干预冠心病)选出980位参与者(230位有心血管疾病事件)的血浆样本进行代谢组学检测,在该研究中使用4种互补的LC-MS检测方法(HILIC-positive(Q Exactive hybrid quadrupole orbitrap mass spectrometer (Thermo Fisher Scientific))、HILIC-negative(5500 QTRAP mass spectrometer [SCIEX]))、C8-positive(Exactive Plus orbitrap mass spectrometer (Thermo Fisher Scientific))、C18-negative(Q Exactive hybrid quadrupole orbitrap mass spec- trometer (Thermo Fisher Scientific))在WHI(-OS/-HT)人群样本中检测到了371个代谢物,其中PREDIMED只使用HILIC-positive、C8-positive两种方法检测。

代谢组学

在WHI-OS人群队列中,通过使用逻辑回归模型发现检测出的371个代谢物中有60个物质与冠心病事件发生显著性相关(以样本筛选匹配的变量为协变量,校正后P<0.05),加上CHD临床风险因子作为协变量发现36个物质与冠心病事件发生显著性相关,17个物质不受CHD临床风险因子影响。在WHI-HT人群队列中验证发现,以样本筛选匹配的变量为协变量有33个物质与冠心病事件发生显著性相关(校正后P<0.05),加上CHD临床风险因子为协变量有8个物质与冠心病事件发生显著性相关,其中7个物质具有增加冠心病发病风险:C34:2 hydroxy-PC, C36:4 hydroxy-PC, 5-hydroxyeicosatetraenoic acid (HETE), 15-HETE, 11-HETE, glutamate, cytidine monophosphate,1个物质具有降低冠心病发病风险:glutamine(如 table2 所示)。

代谢组学

将8个在2个独立人群队列中经过验证的代谢物在逻辑回归模型中进行相互校正,发现只有C34:2 hydroxy-PC在WHI人群中仍然与冠心病事件发生显著性相关,该物质含量最高四分位绝经女性人群比最低四分位绝经女性人群有4.69倍 (95% confidence interval, 2.84– 7.90) 的风险患冠心病。如图1所示根据C34:2 hydroxy-PC含量进行WHI-HT人群队列亚组分型,该物质与冠心病临床风险因子关系仍然显著相关。典型CHD风险因子(ROC曲线下面积AUC=0.76)与C34:2 hydroxy-PC结合(AUC=0.79)进行冠心病预测显著性提高了AUC(95% confidence interval, 0.01–0.05; P<0.01)。

代谢组学
图1. WHI-HT队列中C34:2 hydroxy-PC含量亚组分型分析。BMI indicates body mass index; CI, confidence interval; CRP, C-reactive protein; HDL, high-density lipopro- tein; hydroxy-PC, hydroxy-phospha- tidylcholine; OR, odds ratio; and WHI-HT, Women’s Health Initiative- Hormone Trials.

为了验证WHI人群队列发现的代谢物与冠心病事件的关联是否具有普适性,使用2种LC-MS检测方法在PREDIMED人群队列中验证了4个物质(glutamine, glutamate, C34:2 hydroxy-PC, and C36:4 hydroxy-PC)与冠心病事件发生的相关性。研究发现:1)除了glutamine物质,其他3个物质在男女混合冠心病事件发生中具有显著性关联且在女性中关联更显著;2)只有glutamate单独在男性冠心病事件发生中显著关联;3)Wald test检验发现性别与hydroxy-PCs间的相互作用显著 (P for interaction <0.001)。

代谢组学

此外,如图2所示,将这8个代谢物与心血管疾病临床标记物等临床指标进行关联分析意外发现,氧化性脂类(C34:2 hydroxy-PC, C36:4 hydroxy-PC, 5-HETE, 15-HETE, and 11-HETE)与 CRP、LPA、Lipoprotein-associated phospholipase A2 activity MASS 指标无显著性相关性。

代谢组学
图2. 代谢物与心血管疾病标记物等临床指标间的斯皮尔曼相关性分析。Left, Spearman correlations are shown on the bottom and confidence interval on the top. Right, Heat map. Both figures are ordered by using hierarchical clustering. ACT indicates Lp-PLA2 activity; BMI, body mass index; BNP, N-terminal pro- brain natriuretic peptide; CMP, cytidine monophosphate; CRP, log C-reactive protein; HETE, hydroxyeicosatetraenoic acid; HDL, high-density lipoprotein; LPA, lipoprotein(a); MASS, Lipoprotein-associated phospholipase A2 mass; SBP, systolic blood pressure; and TCHL, total cholesterol.

为了研究371个代谢物间的相关性,使用偏相关分析校正“是否患冠心病”这个协变量,以 |偏相关系数| >0.7为条件,发现有6个cluster物质聚类间的关联与冠心病相关,如图3所示的一个cluster包含了氧化性脂质,且都是上述研究发现的与冠心病事件发生显著性关联的2 个hydroxy-PCs 和4 个HETEs,该cluster与冠心病事件发生显著性相关(P=3.185E-07) 。在该cluster中,以CHD临床风险因子为协变量校正发现有3个物质仍然与冠心病事件发生显著性相关,以样本筛选匹配的变量为协变量校正发现只有1个物质(12-HETE)与冠心病事件发生显著性相关(校正后P<0.05)。

代谢组学
图3. 六个氧化脂类间的偏相关斯皮尔曼相关性分析。Enrichment P value=3.185E-07. All markers are positively associated with increased CHD risk. All correlations (edges) are positive. CHD indicates coronary heart disease; HETE, hydroxyeicosatetraenoic acid; OH-PC, hydroxy-phosphatidyl- choline; and OR, odds ratio. 

综上所述,通过对WHI-OS和WHI-HT两个人群队列中绝经女性的血浆代谢组学进行相互的独立验证研究,发现新的与冠心病事件发生显著性相关的代谢物(如hydroxy-PCs,HETEs,glutamate,glutamine),且在PREDIMED人群队列中再次验证表明C34:2 hydroxy-PC物质与性别和冠心病事件发生具有显著性关联,这些结果为氧化脂质与冠心病的相互关联提供了数据支持和潜在靶点。

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代谢组学