Mangelis Anastasios, Dieterich Peter, Peitzsch Mirko, Richter Susan, Jühlen Ramona, Hübner Angela, Willenberg Holger S, Deussen Andreas, Lenders Jacques W M, Eisenhofer Graeme
Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Fetscherstraße 74, 01307 Dresden, Germany.
Institute of Physiology, University Hospital Carl Gustav Carus, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Fetscherstraße 74, 01307 Dresden, Germany.
J Steroid Biochem Mol Biol. 2016 Jan;155(Pt A):67-75. doi: 10.1016/j.jsbmb.2015.09.038. Epub 2015 Oct 3.
Adrenal steroid hormones, which regulate a plethora of physiological functions, are produced via tightly controlled pathways. Investigations of these pathways, based on experimental data, can be facilitated by computational modeling for calculations of metabolic rate alterations. We therefore used a model system, based on mass balance and mass reaction equations, to kinetically evaluate adrenal steroidogenesis in human adrenal cortex-derived NCI H295R cells. For this purpose a panel of 10 steroids was measured by liquid chromatographic-tandem mass spectrometry. Time-dependent changes in cell incubate concentrations of steroids - including cortisol, aldosterone, dehydroepiandrosterone and their precursors - were measured after incubation with angiotensin II, forskolin and abiraterone. Model parameters were estimated based on experimental data using weighted least square fitting. Time-dependent angiotensin II- and forskolin-induced changes were observed for incubate concentrations of precursor steroids with peaks that preceded maximal increases in aldosterone and cortisol. Inhibition of 17-alpha-hydroxylase/17,20-lyase with abiraterone resulted in increases in upstream precursor steroids and decreases in downstream products. Derived model parameters, including rate constants of enzymatic processes, appropriately quantified observed and expected changes in metabolic pathways at multiple conversion steps. Our data demonstrate limitations of single time point measurements and the importance of assessing pathway dynamics in studies of adrenal cortical cell line steroidogenesis. Our analysis provides a framework for evaluation of steroidogenesis in adrenal cortical cell culture systems and demonstrates that computational modeling-derived estimates of kinetic parameters are an effective tool for describing perturbations in associated metabolic pathways.
肾上腺类固醇激素可调节多种生理功能，其产生过程经由严格控制的途径。基于实验数据对这些途径进行研究时，可通过计算模型来辅助计算代谢率变化。因此，我们使用了一个基于质量平衡和质量反应方程的模型系统，对源自人肾上腺皮质的NCI H295R细胞中的肾上腺类固醇生成进行动力学评估。为此，通过液相色谱 - 串联质谱法测定了一组10种类固醇。在用血管紧张素II、福斯高林和阿比特龙孵育后，测量了类固醇（包括皮质醇、醛固酮、脱氢表雄酮及其前体）在细胞培养液中的浓度随时间的变化。使用加权最小二乘法拟合，根据实验数据估计模型参数。观察到血管紧张素II和福斯高林诱导的培养液中前体类固醇浓度随时间的变化，其峰值先于醛固酮和皮质醇的最大增加。用阿比特龙抑制17α - 羟化酶/17,20 - 裂解酶会导致上游前体类固醇增加，下游产物减少。推导得到的模型参数，包括酶促过程的速率常数，在多个转化步骤中恰当地量化了代谢途径中观察到的和预期的变化。我们的数据证明了单时间点测量的局限性以及在肾上腺皮质细胞系类固醇生成研究中评估途径动力学的重要性。我们的分析为评估肾上腺皮质细胞培养系统中的类固醇生成提供了一个框架，并表明通过计算模型推导的动力学参数估计是描述相关代谢途径扰动的有效工具。
