Combined liquid and solid-phase extraction improves quantification of brain estrogen content.
Combined liquid and solid-phase extraction improves quantification of brain estrogen content.
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DOI：
10.3389/fnana.2011.00057
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发表时间：
2011
影响因子：
2.9
通讯作者：
Remage-Healey L
中科院分区：
文献类型：
--
作者：
Chao A;Schlinger BA;Remage-Healey L
定量脑源性类固醇激素（“神经类固醇”）的准确性对于理解神经元活动、发育和生理学的调节变得越来越重要。相对于其他神经活性化合物和经典的神经递质，类固醇由于其脂溶性而在分离和分析方面提出了特别的挑战。因此，神经甾体分布的解剖学研究主要依赖于神经甾体合成酶的表达。为了评估合成酶维斯维斯实际类固醇本身的分布，传统的类固醇定量测定，包括放射免疫测定，已经成功地采用了液体提取方法（例如，乙醚、二氯甲烷或甲醇）从显微解剖的脑组织中分离类固醇。由于其灵敏度，安全性和可靠性，使用商业酶免疫测定（EIA）的血浆和大脑中的类固醇的实验室定量已变得越来越普遍。然而，EIA依赖于体外酶反应，使其对脑组织中的干扰物质敏感，从而产生不可靠的结果。在这里，我们评估的有效性相结合，两阶段的液体/固相萃取（SPE）相比，传统的液体提取单独的雌二醇（E2）从脑组织中的分离协议。我们采用鸣禽模型系统，其中大脑类固醇的产生是明显的，并与学习和可塑性的神经机制。这项研究概述了一个组合的液体SPE协议，提高了性能的商业EIA定量脑E2含量。我们证明了我们的优化方法用于评估脑E2含量的区域特异性的有效性，将这些结果与雌激素合成酶和雌激素受体的已建立解剖学进行比较，并讨论潜在EIA干扰物质的性质。
Accuracy in quantifying brain-derived steroid hormones (“neurosteroids”) has become increasingly important for understanding the modulation of neuronal activity, development, and physiology. Relative to other neuroactive compounds and classical neurotransmitters, steroids pose particular challenges with regard to isolation and analysis, owing to their lipid solubility. Consequently, anatomical studies of the distribution of neurosteroids have relied primarily on the expression of neurosteroid synthesis enzymes. To evaluate the distribution of synthesis enzymes vis-à-vis the actual steroids themselves, traditional steroid quantification assays, including radioimmunoassays, have successfully employed liquid extraction methods (e.g., ether, dichloromethane, or methanol) to isolate steroids from microdissected brain tissue. Due to their sensitivity, safety, and reliability, the use of commercial enzyme-immunoassays (EIA) for laboratory quantification of steroids in plasma and brain has become increasingly widespread. However, EIAs rely on enzymatic reactions in vitro, making them sensitive to interfering substances in brain tissue and thus producing unreliable results. Here, we evaluate the effectiveness of a protocol for combined, two-stage liquid/solid-phase extraction (SPE) as compared to conventional liquid extraction alone for the isolation of estradiol (E2) from brain tissue. We employ the songbird model system, in which brain steroid production is pronounced and linked to neural mechanisms of learning and plasticity. This study outlines a combined liquid–SPE protocol that improves the performance of a commercial EIA for the quantification of brain E2 content. We demonstrate the effectiveness of our optimized method for evaluating the region specificity of brain E2 content, compare these results to established anatomy of the estrogen synthesis enzyme and estrogen receptor, and discuss the nature of potential EIA interfering substances.
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影响因子：
2.8
作者：
Remage-Healey L;London SE;Schlinger BA
通讯作者：
Schlinger BA
影响因子：
3.2
作者：
Remage-Healey L;Oyama RK;Schlinger BA
通讯作者：
Schlinger BA
影响因子：
4.8
作者：
LARNER, JM;ROSNER, W;HOCHBERG, RB
通讯作者：
HOCHBERG, RB
DOI：
10.1086/physzool.60.2.30158643
发表时间：
1987-03-01
期刊：
PHYSIOLOGICAL ZOOLOGY
影响因子：
--
作者：
BALL, GF;WINGFIELD, JC
通讯作者：
WINGFIELD, JC
影响因子：
4.8
作者：
London, Sarah E.;Monks, D. Ashley;Schlinger, Barney A.
通讯作者：
Schlinger, Barney A.