Wojnicz Aneta, Avendaño-Ortiz José, de Pascual Ricardo, Ruiz-Pascual Lucía, García Antonio G, Ruiz-Nuño Ana
Instituto-Fundación Teófilo Hernando, Universidad Autónoma de Madrid, Madrid, Spain.
Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, Spain.
J Mass Spectrom. 2016 Aug;51(8):651-664. doi: 10.1002/jms.3794.
The primary functions of adrenal medullary chromaffin cells are the synthesis and storage in their chromaffin vesicles of the catecholamines noradrenaline (NA) and adrenaline (AD), and their subsequent release into the bloodstream by Ca -dependent exocytosis under conditions of fear or stress (fight or flight response). Several monoamines, nucleotides and opiates, such as leucine-enkephalin (LENK) and methionine-enkephalin (MENK), are also co-stored and co-released with the catecholamines. However, other neurotransmitters have not been studied in depth. Here, we present a novel high-resolution liquid chromatography-tandem mass spectrometry approach for the simultaneous monitoring of 14 compounds stored and released in bovine chromaffin cells (BCCs). We validated the analytical method according to the recommendations of the EMA and FDA by testing matrix effect, selectivity, sensitivity, precision, accuracy, stability and carry-over. After testing on six batches of BCCs from different cultures, the method enabled simultaneous quantitative determination of monoamines (AD, NA, dopamine, serotonin, 5-hydroxyindoleacetic acid, histamine and metanephrine), amino acids (L-glutamic acid, γ-aminobutyric acid), nucleotides (adenosine 5'-diphosphate, adenosine 5'-monophosphate, cyclic adenosine 5'-monophosphate) and neuropeptides (LENK and MENK) in the intracellular content, basal secretion and acetylcholine induced secretion of BBCs. The high-resolution approach used here enabled us to determine the levels of 14 compounds in the same BCC batch in only 16 min. This novel approach will make it possible to study the regulatory mechanisms of Ca signaling, exocytosis and endocytosis using different neurotrophic factors and/or secretagogues as stimuli in primary BCC cultures. Our method is actually being applied to human plasma samples of different therapeutic areas where sympathoadrenal axis is involved in stress situations such as Alzheimer's disease, migraine or cirrhosis, to improve diagnosis and clinical practice. Copyright © 2016 John Wiley & Sons, Ltd.
肾上腺髓质嗜铬细胞的主要功能是在其嗜铬小泡中合成并储存儿茶酚胺类物质去甲肾上腺素（NA）和肾上腺素（AD），并在恐惧或应激状态（战斗或逃跑反应）下通过钙依赖性胞吐作用将它们释放到血液中。几种单胺、核苷酸和阿片类物质，如亮氨酸脑啡肽（LENK）和甲硫氨酸脑啡肽（MENK），也与儿茶酚胺类物质共同储存并共同释放。然而，其他神经递质尚未得到深入研究。在此，我们提出了一种新颖的高分辨率液相色谱 - 串联质谱方法，用于同时监测牛嗜铬细胞（BCCs）中储存和释放的14种化合物。我们根据欧洲药品管理局（EMA）和美国食品药品监督管理局（FDA）的建议，通过测试基质效应、选择性、灵敏度、精密度、准确度、稳定性和残留，验证了该分析方法。在对来自不同培养物的六批BCCs进行测试后，该方法能够同时定量测定细胞内含量、基础分泌以及乙酰胆碱诱导分泌的BCCs中的单胺类物质（AD、NA、多巴胺、血清素、5 - 羟基吲哚乙酸、组胺和间甲肾上腺素）、氨基酸（L - 谷氨酸、γ - 氨基丁酸）、核苷酸（腺苷5'-二磷酸、腺苷5'-单磷酸、环腺苷5'-单磷酸）和神经肽（LENK和MENK）。这里使用的高分辨率方法使我们能够在仅16分钟内测定同一批BCCs中14种化合物的水平。这种新颖的方法将有可能在原代BCC培养物中使用不同的神经营养因子和/或促分泌剂作为刺激物来研究钙信号传导、胞吐作用和内吞作用的调节机制。我们的方法实际上正在应用于涉及交感肾上腺轴的不同治疗领域的人体血浆样本，如阿尔茨海默病、偏头痛或肝硬化等应激情况，以改善诊断和临床实践。版权所有© 2016约翰威立父子有限公司。
