Simultaneous monitoring of monoamines, amino acids, nucleotides and neuropeptides by liquid chromatography-tandem mass spectrometry and its application to neurosecretion in bovine chromaffin cells

Abstract

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.

Metabolomics Analyses of 14 Classical Neurotransmitters by GC-TOF with LC-MS Illustrates Secretion of 9 Cell–Cell Signaling Molecules from Sympathoadrenal Chromaffin Cells in the Presence of Lithium

“…Therefore, this study developed targeted MS identification and quantitation of 14 classical neurotransmitters by GC-TOF (gas chromatography time-of-flight), combined with LC-MS/MS (liquid chromatography coupled to tandem mass spectrometry) analyses. GC-TOF is appropriate for analyses of low molecular weight molecules , but has not been used in prior neurotransmitter analyses, which have largely utilized LC-MS and LC-MS/MS. − . Results of this study found that GC-TOF, combined with LC-MS/MS, provides an improved metabolomics strategy for analyses of 14 classical neurotransmitters consisting of acetylcholine, adenosine, anandamide, aspartate, dopamine, epinephrine, GABA, glutamate, glycine, histamine, melatonin, norepinephrine, serine, and serotonin.…”

“…However, to the best of our knowledge based on extensive literature evaluation, there are currently no publications that have reported identification and quantitation of the major spectrum of 14 classical neurotransmitters, as analyzed by this study. While many studies have conducted MS analyses of “neurotransmitters”, most have only analyzed 3–6 neurotransmitters − and a few have analyzed 7–8 neurotransmitters. − Many of these studies of “neurotransmitters” include metabolites, rather than only the bona fide neurotransmitters. For these reasons, there is a need to develop metabolomics methodology for quantitative profiling of the major repertoire of 14 classical neurotransmitter molecules, the goal of this study.…”

The Adrenal Medulla Modulates Mechanical Allodynia in a Rat Model of Neuropathic Pain

“…The supernatant was collected, centrifuged at 3000 rpm for 2 min at 4 • C (Eppendorf, Germany), and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A system consisting of a 1200 Series Liquid Chromatograph coupled to a 6410B Triple Quadrupole Mass Spectrometer (LC-MS; Agilent Technologies, Madrid, Spain) was used [57], and CAs were determined using a positive mode electrospray ionization; likewise, the pellet was analyzed for protein content.…”

Role of serotonin in the paracrine control of adrenal steroidogenesis in physiological and pathophysiological conditions

“…The presence of 5-HT in adrenal glands has been demonstrated in various species including fish, frog, mouse, rat, pig, cattle and human [15][16][17][18][19][20][21]. The cell types that contain 5-HT differ among species.…”