通过 LC-MS/MS 对醛固酮、皮质醇和可的松进行快速可靠的定量,以研究原代细胞培养物中 11β- 羟类固醇脱氢酶的活性。

IF 4.6 Q2 MATERIALS SCIENCE, BIOMATERIALS
Sonja Kunz , Yao Meng , Holger Schneider , Laura Brunnenkant , Michaela Höhne , Tim Kühnle , Martin Reincke , Marily Theodoropoulou , Martin Bidlingmaier
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引用次数: 0

摘要

细胞培养实验有助于确定健康和肿瘤人体组织中酶活性的特征。液相色谱-串联质谱法(LC-MS/MS)可同时测定单个样品中的多种类固醇,有助于分析类固醇生物合成的分子途径。我们开发了一种可靠而快速的方法,用于定量检测细胞培养上清液中的皮质醇、可的松和醛固酮。我们对两种不同类型的细胞进行了验证,包括基质匹配校准调查。在研究不同类型细胞中糖皮质激素和矿质皮质激素过量条件下的 11β- 羟类固醇脱氢酶 2 型(HSD11B2)活性时,证明了该方法的实用性。用甲基叔丁基醚液液萃取法(LLE)从 1 毫升细胞培养上清液中提取醛固酮、皮质醇和可的松。类固醇经 Kinetex 联苯柱(50 ×2.1mm,2.6µm)分离,水和甲醇(含 2mM 氨格式)梯度洗脱,电喷雾正离子后在多反应监测模式下进行分析。该方法的应用包括两种不同原代细胞类型的细胞培养实验,即人冠状动脉平滑肌细胞(HCSMC)和人冠状动脉内皮细胞(EC)。用不同浓度的皮质醇、醛固酮和米非司酮(一种糖皮质激素受体拮抗剂)处理细胞,然后进行定量 PCR 分析。该方法的精密度(CV ≤ 6%)和准确度(与标称浓度的偏差 ≤ 6%)都很高,醛固酮、可的松和皮质醇的定量限(LoQ)分别为 0.11、0.56 和 0.69 nmol/L。在介质或溶剂中制备的校准曲线没有差异。通过该方法,我们确认了 HSD11B2 的活性以及皮质醇在 HCSMC 中转化为可的松的浓度依赖性(140nM 皮质醇时的中位转化率 = 1.46%)。相比之下,我们在 EC 中没有观察到任何 HSD11B2 活性。添加高浓度醛固酮或添加 1µM 米非司酮都不会影响糖皮质激素的浓度。定量 PCR 发现 HCSMC 中有 HSD11B1 和 HSD11B2 的表达,而 EC 中没有。我们提出了一种快速可靠的方法来定量检测细胞培养上清液中的皮质醇、可的松和醛固酮。该方法使我们能够研究两种不同类型细胞中 HSD11B2 的活性,并将支持未来研究糖皮质激素和矿质类固醇过量条件下靶器官损伤机制的实验。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Fast and reliable quantification of aldosterone, cortisol and cortisone via LC-MS/MS to study 11β-hydroxysteroid dehydrogenase activities in primary cell cultures

Cell culture experiments can support characterization of enzymatic activities in healthy and tumorous human tissues. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) enables simultaneous measurement of several steroids from a single sample, facilitating analysis of molecular pathways involved in steroid biosynthesis. We developed a reliable but fast method for quantification of cortisol, cortisone and aldosterone in cell culture supernatant. Validation, including investigation of matrix-matched calibration, was performed for two different cell types. Utility of the method was demonstrated in the study of 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) activity under conditions of glucocorticoid and mineralocorticoid excess in different cell types. Aldosterone, cortisol and cortisone were extracted by liquid-liquid extraction (LLE) with methyl tert-butyl ether from 1 mL of cell culture supernatant. Steroids were separated on a Kinetex biphenyl column (50 ×2.1 mm, 2.6 µm) with gradient elution of water and methanol containing 2 mM ammonium format and analysed in multiple reaction monitoring mode after positive electrospray ionization. Application of the method included cell culture experiments with two different primary cell types, human coronary artery smooth muscle cells (HCSMC) and human coronary artery endothelial cells (EC). Cells were treated with different concentrations of cortisol, aldosterone and mifepristone, a glucocorticoid receptor antagonist and quantitative PCR was performed. The method exhibits high precision (CV ≤ 6 %) and accuracy (deviation from nominal concentration ≤ 6 %) for concentrations above the limit of quantification (LoQ) which is 0.11, 0.56 and 0.69 nmol/L for aldosterone, cortisone and cortisol, respectively. Calibration curves did not differ when prepared in media or solvent. The method enabled us to confirm activity of HSD11B2 and concentration dependent conversion of cortisol to cortisone in HCSMC (median conversion ratio at 140 nM cortisol = 1.46 %). In contrast we did not observe any HSD11B2 activity in EC. Neither addition of high aldosterone, nor addition of 1 µM mifepristone had impact on glucocorticoid concentrations. Quantitative PCR revealed expression of HSD11B1 and HSD11B2 in HCSMC but not in EC. We present a fast and reliable method for quantification of cortisol, cortisone and aldosterone in cell culture supernatants. The method enabled us to study HSD11B2 activity in two different cell types and will support future experiments investigating mechanisms of target organ damage in conditions of glucocorticoid and mineralocorticoid excess.

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来源期刊
ACS Applied Bio Materials
ACS Applied Bio Materials Chemistry-Chemistry (all)
CiteScore
9.40
自引率
2.10%
发文量
464
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