Simultaneous quantification of monoamine neurotransmitters and their biogenic metabolites intracellularly and extracellularly in primary neuronal cell cultures and in sub-regions of guinea pig brain

Schou-Pedersen, Anne Marie V.; Hansen, Stine Normann; Tveden‐Nyborg, Pernille; Lykkesfeldt, Jens

doi:10.1016/j.jchromb.2016.05.048

Cited by 15 publications

(12 citation statements)

References 30 publications

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“…14 Therefore, the simultaneous determination of catecholamines and their metabolites including the two endproducts would allow us to better understand the overall brain homeostasis. Although an ECD-HPLC assay for DA, NE, and E, 15 and an immunoassay for HVA as inflammatory marker 16 have been proposed so far, no reports have been published on a simultaneous assay for all six metabolites due to the poor separation capacities of common reversed-phase LC columns.…”

Section: Resultsmentioning

confidence: 99%

Analysis of Catecholamine and Their Metabolites in Mice Brain by Liquid Chromatography-Mass Spectrometry Using Sulfonated Mixed-mode Copolymer Column

et al. 2018

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In this study, a simultaneous assay for catecholamines and their metabolites in the brain was established using liquid chromatography-mass spectrometry (LC-MS). To achieve complete separation, a cation-exchange/reversed-phase mixedmode copolymer resin column containing 0.81 wt% sulfo groups was used for the simultaneous LC-MS assay. The analyzed catecholamines were dopamine (DA), norepinephrine (NE), and epinephrine (E), while the metabolites lacking amino groups were 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 3-methoxy-4hydroxyphenylglycol (MHPG). The metabolites were separated and detected using LC-MS, on columns with and without sulfo groups. However, we could not achieve adequate separation of catecholamines on both columns using a gradient elution of 0 -50 (v/v)% methanol containing 0.1 (v/v)% formic acid (FA). When volatile ion-pairing reagents were added to the mobile phase, they improved the retention and detection of catecholamines on the sulfonated mixed-mode column. Under optimized elution conditions, which involved a linear gradient elution of water containing 0.1 (v/v)% FA to 50 (v/v)% acetonitrile in 50 mM ammonium formate at 40 C and a 0.20 mL/min rate, all six target molecules were simultaneously detected within 25 min, when using negative mode LC-MS on a sulfonated mixed-mode column. The limits of detection (LODs) for DA, NE, E, DOPCA, HVA, and MHPG were determined to be 20. 7, 12.6, 74.6, 1110, 18.7, and 3196 nM, respectively. Moreover, the established LC-MS assay allowed the detection of endogenous DA, NE, and HVA, in normal mouse brain samples at concentrations higher than 20, 9, and 4 pmol/mg, respectively.

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Section: Resultsmentioning

confidence: 99%

Analysis of Catecholamine and Their Metabolites in Mice Brain by Liquid Chromatography-Mass Spectrometry Using Sulfonated Mixed-mode Copolymer Column

et al. 2018

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“…Cortisol in pig plasma was measured enzymatically by ELISA using the DRG Cortisol human ELISA kit (EIA‐1887) 16 with an LLOQ of 55 pmol/L. Plasma levels of norepinephrine and epinephrine were quantified by an HPLC method with electrochemical detection essentially as described elsewhere 17 . The samples were run in duplicate in a randomized order.…”

Section: Methodsmentioning

confidence: 99%

The counterregulatory response to hypoglycaemia in the pig

Gradel

Ludvigsen

Porsgaard

et al. 2020

Basic Clin Pharma Tox

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The domestic pig is commonly used as animal model in the pharmaceutical development of new therapeutics for treatment of diabetes. Since a formal definition of hypoglycaemia only exists in humans, the purpose of this study was to assess the counterregulatory response in the domestic pig at glucose levels known to induce symptoms of hypoglycaemia in humans. Six pigs were included in hyperinsulinaemic glucose clamps with plasma glucose targets of 2, 3 and 5 mmol/L in a cross‐over design, and the associated glucose counterregulatory response was assessed by measuring glucose kinetics and levels of glucagon, c‐peptide, catecholamines, cortisol and growth hormone. Results showed that the 2 and 3 vs 5 mmol/L clamps significantly decreased and increased the secretion of c‐peptide and glucagon, respectively (P < .05). This finding was associated with increased rate of glucose appearance (Ra) and decreased rate of glucose disappearance (Rd) (P < .001). No marked differences in the catecholamine, growth hormone or cortisol response were observed. Consequently, like humans, pigs respond to hypoglycaemia by decreasing the pancreatic output of insulin while increasing that of glucagon, with increased glucose mobilization and decreased glucose disposal as a result. The hypoglycaemic clamps did not result in a marked secretion of the other counterregulatory hormones.

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“…The cell pellet was thawed, and the cells were lysed with an ultrasonic cell disruptor after the addition of 2 mL water containing 0.2% formic acid, followed by precipitation and centrifugation. When analyzed, 20 μL internal standard solution was added to 200 μL of cell supernatant [ 27 ].…”

Section: Methodsmentioning

confidence: 99%

Integrated Proteomics and Lipidomics Investigation of the Mechanism Underlying the Neuroprotective Effect of N-benzylhexadecanamide

et al. 2018

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Macamides are very important secondary metabolites produced by Lepidium meyenii Walp, which possess multiple bioactivities, especially in the neuronal system. In a previous study, we observed that macamides exhibited excellent effects in the recovery of injured nerves after 1-methyl-4-phenylpyridinium (MPP+)-induced dopaminergic neuronal damage in zebrafish. However, the mechanism underlying this effect remains unclear. In the present study, we observed that N-benzylhexadecanamide (XA), which is a typical constituent of macamides, improved the survival rate of neurons in vitro. We determined the concentration of neurotransmitters in MN9D cells and used it in conjunction with an integrated proteomics and lipidomics approach to investigate the mechanism underlying the neuroprotective effects of XA in an MPP+-induced neurodegeneration cell model using QqQ MS, Q-TOF MS, and Orbitrap MS. The statistical analysis of the results led to the identification of differentially-expressed biomarkers, including 11 proteins and 22 lipids, which may be responsible for the neuron-related activities of XA. All these potential biomarkers were closely related to the pathogenesis of neurodegenerative diseases, and their levels approached those in the normal group after treatment with XA. Furthermore, seven lipids, including five phosphatidylcholines, one lysophosphatidylcholine, and one phosphatidylethanolamine, were verified by a relative quantitative approach. Moreover, four proteins (Scarb2, Csnk2a2, Vti1b, and Bnip2) were validated by ELISA. The neurotransmitters taurine and norepinephrine, and the cholinergic constituents, correlated closely with the neuroprotective effects of XA. Finally, the protein–lipid interaction network was analyzed. Based on our results, the regulation of sphingolipid metabolism and mitochondrial function were determined to be the main mechanisms underlying the neuroprotective effect of XA. The present study should help us to better understand the multiple effects of macamides and their use in neurodegenerative diseases.

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Simultaneous quantification of monoamine neurotransmitters and their biogenic metabolites intracellularly and extracellularly in primary neuronal cell cultures and in sub-regions of guinea pig brain

Cited by 15 publications

References 30 publications

Analysis of Catecholamine and Their Metabolites in Mice Brain by Liquid Chromatography-Mass Spectrometry Using Sulfonated Mixed-mode Copolymer Column

Analysis of Catecholamine and Their Metabolites in Mice Brain by Liquid Chromatography-Mass Spectrometry Using Sulfonated Mixed-mode Copolymer Column

The counterregulatory response to hypoglycaemia in the pig

Integrated Proteomics and Lipidomics Investigation of the Mechanism Underlying the Neuroprotective Effect of N-benzylhexadecanamide

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