TY - JOUR A1 - Schumacher, Fabian A1 - Chakraborty, Sudipta A1 - Kleuser, Burkhard A1 - Gulbins, Erich A1 - Schwerdtle, Tanja A1 - Aschner, Michael A. A1 - Bornhorst, Julia T1 - Highly sensitive isotope-dilution liquid-chromatography-electrospray ionization-tandem-mass spectrometry approach to study the drug-mediated modulation of dopamine and serotonin levels in Caenorhabditis elegans JF - Talanta : the international journal of pure and applied analytical chemistry N2 - Dopamine (DA) and serotonin (SRT) are monoamine neurotransmitters that play a key role in regulating the central and peripheral nervous system. Their impaired metabolism has been implicated in several neurological disorders, such as Parkinson's disease and depression. Consequently, it is imperative to monitor changes in levels of these low-abundant neurotransmitters and their role in mediating disease. For the first time, a rapid, specific and sensitive isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of DA and SRT in the nematode Caenorhabditis elegans (C. elegans). This model organism offers a unique approach for studying the effect of various drugs and environmental conditions on neurotransmitter levels, given by the conserved DA and SRT biology, including synaptic release, trafficking and formation. We introduce a novel sample preparation protocol incorporating the usage of sodium thiosulfate in perchloric acid as extraction medium that assures high recovery of the relatively unstable neurotransmitters monitored. Moreover, the use of both deuterated internal standards and the multiple reaction monitoring (MRM) technique allows for unequivocal quantification. Thereby, to the best of our knowledge, we achieve a detection sensitivity that clearly exceeds those of published DA and SRT quantification methods in various matrices. We are the first to show that exposure of C elegans to the monoamine oxidase B (MAOB) inhibitor selegiline or the catechol-O-methyltransferase (COMT) inhibitor tolcapone, in order to block DA and SRT degradation, resulted in accumulation of the respective neurotransmitter. Assessment of a behavioral output of the dopaminergic system (basal slowing response) corroborated the analytical LC-MS/MS data. Thus, utilization of the C elegans model system in conjunction with our analytical method is well-suited to investigate drug-mediated modulation of the DA and SRT system in order to identify compounds with neuroprotective or regenerative properties. (C) 2015 Elsevier B.V. All rights reserved. KW - Caenorhabditis elegans KW - Dopamine KW - Serotonin KW - Liquid chromatography-tandem mass spectrometry KW - Isotope-dilution analysis Y1 - 2015 U6 - https://doi.org/10.1016/j.talanta.2015.05.057 SN - 0039-9140 SN - 1873-3573 VL - 144 SP - 71 EP - 79 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Neuber, Corinna A1 - Schumacher, Fabian A1 - Gulbins, Erich A1 - Kleuser, Burkhard T1 - Method to simultaneously determine the sphingosine 1-phosphate breakdown product (2E)-hexadecenal and its fatty acid derivatives using isotope-dilution HPLC-electrospray ionization-quadrupole/time-of-flight mass spectrometry JF - Analytical chemistry N2 - Sphingosine 1-phosphate (S1P), a bioactive lipid involved in various physiological processes, can be irreversibly degraded by the membrane-bound S1P lyase (S1PL) yielding (2E)-hexadecenal and phosphoethanolamine. It is discussed that (2E)-hexadecenal is further oxidized to (2E)-hexadecenoic acid by the long-chain fatty aldehyde dehydrogenase ALDH3A2 (also known as FALDH) prior to activation via coupling to coenzyme A (CoA). Inhibition or defects in these enzymes, S1PL or FALDH, result in severe immunological disorders or the Sjogren-Larsson syndrome, respectively. Hence, it is of enormous importance to simultaneously determine the S1P breakdown product (2E)-hexadecenal and its fatty acid metabolites in biological samples. However, no method is available so far. Here, we present a sensitive and selective isotope-dilution high performance liquid chromatographyelectrospray ionizationquadrupole/time-of-flight mass spectrometry method for simultaneous quantification of (2E)-hexadecenal and its fatty acid metabolites following derivatization with 2-diphenylacetyl-1,3-indandione-1-hydrazone and 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide. Optimized conditions for sample derivatization, chromatographic separation, and MS/MS detection are presented as well as an extensive method validation. Finally, our method was successfully applied to biological samples. We found that (2E)-hexadecenal is almost quantitatively oxidized to (2E)-hexadecenoic acid, that is further activated as verified by cotreatment of HepG2 cell lysates with (2E)-hexadecenal and the acyl-CoA synthetase inhibitor triacsin C. Moreover, incubations of cell lysates with deuterated (2E)-hexadecenal revealed that no hexadecanoic acid is formed from the aldehyde. Thus, our method provides new insights into the sphingolipid metabolism and will be useful to investigate diseases known for abnormalities in long-chain fatty acid metabolism, e.g., the Sjogren-Larsson syndrome, in more detail. Y1 - 2014 U6 - https://doi.org/10.1021/ac501677y SN - 0003-2700 SN - 1520-6882 VL - 86 IS - 18 SP - 9065 EP - 9073 PB - American Chemical Society CY - Washington ER - TY - GEN A1 - Chakraborty, Sudipta A1 - Chen, Pan A1 - Bornhorst, Julia A1 - Schwerdtle, Tanja A1 - Schumacher, Fabian A1 - Kleuser, Burkhard A1 - Bowman, Aaron B. A1 - Aschner, Michael A. T1 - Loss of pdr-1/parkin influences Mn homeostasis through altered ferroportin expression in C. elegans N2 - Overexposure to the essential metal manganese (Mn) can result in an irreversible condition known as manganism that shares similar pathophysiology with Parkinson's disease (PD), including dopaminergic (DAergic) cell loss that leads to motor and cognitive impairments. However, the mechanisms behind this neurotoxicity and its relationship with PD remain unclear. Many genes confer risk for autosomal recessive, early-onset PD, including the parkin/PARK2 gene that encodes for the E3 ubiquitin ligase Parkin. Using Caenorhabditis elegans (C. elegans) as an invertebrate model that conserves the DAergic system, we previously reported significantly increased Mn accumulation in pdr-1/parkin mutants compared to wildtype (WT) animals. For the current study, we hypothesize that this enhanced accumulation is due to alterations in Mn transport in the pdr-1 mutants. While no change in mRNA expression of the major Mn importer proteins (smf-1-3) was found in pdr-1 mutants, significant downregulation in mRNA levels of the putative Mn exporter ferroportin (fpn-1.1) was observed. Using a strain overexpressing fpn-1.1 in worms lacking pdr-1, we show evidence for attenuation of several endpoints of Mn-induced toxicity, including survival, metal accumulation, mitochondrial copy number and DAergic integrity, compared to pdr-1 mutants alone. These changes suggest a novel role of pdr-1 in modulating Mn export through altered transporter expression, and provides further support of metal dyshomeostasis as a component of Parkinsonism pathophysiology. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 290 Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-99508 ER -