Laboratory of Pharmaceutical Analysis, Faculty of medicine and pharmacy, Research Institute for Health Sciences and Technology, University of Mons, Place du Parc 20, 7000 Mons, Belgium.
Metabolomic Diagnostics, Little Island, Cork, Ireland.
Talanta. 2018 Sep 1;187:193-199. doi: 10.1016/j.talanta.2018.05.020. Epub 2018 May 7.
Marinobufagenin (MBG) is a bufadienolide cardiac inotrope implicated in volume expansion-mediated hypertensive states including essential hypertension and preeclampsia (PE). Endogenous MBG is an inhibitor of the α1-isoform of Na,K-ATPase with vasoconstrictive and cardiotonic properties, causing hypertension and natriuresis. Elevated endogenous MBG-like material levels have been described by immunoassays in salt-sensitive pregnant and preeclamptic rats as well as in preeclamptic human patients. The rise of endogenous MBG-like material appears prior the development of the main symptoms of PE, leading us to consider MBG as one of the potential biomarkers for PE. The weak specificity and the high variability of the published immunoassays gives no certification about endogenous MBG existence. This led us to set-up a highly specific and sensitive analytical method to detect MBG in plasma at low levels relying on liquid chromatography combined to mass spectrometry (UHPLC-MS/MS) with recording of 7 highly specific MRM transitions for MBG. Pure MBG standard used in the method development was obtained by purification from the Bufo marinus toad venom. d-25-hydroxyvitamin D3 was used as internal standard. An increasing organic gradient with mobile phase A and B composed of 97:3 (v/v) HO: MeOH and 50:45:5 (v/v/v) MeOH:IPA:HO at pH 4.5 respectively was used on a Pursuit 3 PFP column (100 mm × 3 mm; 3 µm) to allow elution and separation of the plasmatic compounds. Chromatographic analyses of plasma samples were preceded by a precipitation of proteins pretreatment. The developed UHPLC-MS/MS assay has been applied to early-pregnant women plasma samples allowing us to investigate MBG plasma levels. Thanks to the high specificity of the assay we were able to authenticate and certify the presence of endogenous MBG in early-pregnant women plasma with the use of the 7 selected specific mass transitions. These pioneering preliminary results are giving a promising perspective for early preeclampsia risk assessment in pregnant women.
海洋蟾蜍精（MBG）是一种蟾蜍甾烯类强心苷，与包括原发性高血压和子痫前期（PE）在内的容量扩张介导的高血压状态有关。内源性 MBG 是 α1-同工型 Na，K-ATP 酶的抑制剂，具有血管收缩和强心作用，导致高血压和利钠作用。免疫测定法已在盐敏感的妊娠和子痫前期大鼠以及子痫前期的人类患者中描述了内源性 MBG 样物质水平升高。内源性 MBG 样物质的升高似乎先于 PE 的主要症状的发展，这使得我们认为 MBG 是 PE 的潜在生物标志物之一。已发表的免疫测定法的特异性弱和变异性高，不能证明内源性 MBG 的存在。这导致我们建立了一种高度特异性和灵敏的分析方法，以检测血浆中的 MBG 低水平，该方法依赖于液相色谱-质谱联用（UHPLC-MS/MS），并记录 7 种针对 MBG 的高度特异性 MRM 转换。用于方法开发的纯 MBG 标准是通过从 Bufo marinus 蟾蜍毒液中纯化获得的。d-25-羟基维生素 D3 被用作内标。在 pH 值为 4.5 的情况下，使用包含 97:3（v/v）HO:MeOH 和 50:45:5（v/v/v）MeOH:IPA:HO 的移动相 A 和 B 的逐渐增加的有机梯度在 Pursuit 3 PFP 柱（100 mm×3 mm；3 µm）上进行，以允许洗脱和分离血浆化合物。在进行蛋白质沉淀预处理之前，对血浆样品进行了色谱分析。所开发的 UHPLC-MS/MS 测定法已应用于早孕妇女的血浆样品，使我们能够研究 MBG 的血浆水平。由于该测定法的特异性高，我们能够使用 7 种选定的特异性质量转换来鉴定和证明内源性 MBG 在早孕妇女血浆中的存在。这些开创性的初步结果为孕妇子痫前期的早期风险评估提供了有希望的前景。
