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首页> 外文期刊>Journal of mass spectrometry: JMS >Simultaneous monitoring of monoamines, amino acids, nucleotides and neuropeptides by liquid chromatography-tandem mass spectrometry and its application to neurosecretion in bovine chromaffin cells
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Simultaneous monitoring of monoamines, amino acids, nucleotides and neuropeptides by liquid chromatography-tandem mass spectrometry and its application to neurosecretion in bovine chromaffin cells

机译：液相色谱-串联质谱法同时监测单胺，氨基酸，核苷酸和神经肽及其在牛嗜铬细胞神经分泌中的应用

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摘要

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 Ca2+-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, gamma-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 16min. This novel approach will make it possible to study the regulatory mechanisms of Ca2+ 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 (C) 2016 John Wiley & Sons, Ltd.
机译：肾上腺髓质嗜铬细胞的主要功能是儿茶酚胺去甲肾上腺素（NA）和肾上腺素（AD）的合成和储存在它们的嗜铬囊泡中，随后在恐惧或压力（战斗）条件下通过依赖Ca2 +的胞吐作用释放入血流或航班响应）。几种单胺，核苷酸和鸦片剂，例如亮氨酸-脑啡肽（LENK）和蛋氨酸-脑啡肽（MENK），也与儿茶酚胺共存和共释放。但是，尚未深入研究其他神经递质。在这里，我们提出了一种新颖的高分辨率液相色谱-串联质谱方法，用于同时监测在牛嗜铬细胞（BCC）中存储和释放的14种化合物。我们通过测试基质效应，选择性，灵敏度，精密度，准确性，稳定性和残留物，根据EMA和FDA的建议对分析方法进行了验证。在对六批不同培养物中的BCC进行测试后，该方法能够同时定量测定单胺（AD，NA，多巴胺，5-羟色胺，5-羟基吲哚乙酸，组胺和间肾上腺素），氨基酸（L-谷氨酸，γ-氨基丁酸） ），细胞内含量，碱基分泌和乙酰胆碱诱导的BBC分泌的核苷酸（腺苷5'-二磷酸，腺苷5'-单磷酸，环状腺苷5'-单磷酸）和神经肽（LENK和MENK）。此处使用的高分辨率方法使我们能够在16分钟内确定同一批BCC批次中14种化合物的水平。这种新颖的方法将有可能研究在原始BCC培养物中使用不同的神经营养因子和/或促分泌素作为刺激物的Ca2 +信号传导，胞吐作用和内吞作用的调节机制。我们的方法实际上已应用于交感肾上腺轴涉及阿尔茨海默氏病，偏头痛或肝硬化等应激情况的不同治疗区域的人血浆样品，以改善诊断和临床实践。版权所有（C）2016 John Wiley＆Sons，Ltd.

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