Structural elucidation
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Specific diagnostic markers are the key to effective diagnosis and treatment of inborn errors of metabolism (IEM). Untargeted metabolomics allows for the identification of potential novel diagnostic biomarkers. Current separation techniques coupled to high-resolution mass spectrometry provide a powerful tool for structural elucidation of unknown compounds in complex biological matrices. This is a proof-of-concept study testing this methodology to determine the molecular structure of as yet uncharacterized m/z signals that were significantly increased in plasma samples from patients with phenylketonuria and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency. A hybrid linear ion trap-orbitrap high resolution mass spectrometer, capable of multistage fragmentation, was used to acquire accurate masses and product ion spectra of the uncharacterized m/z signals. In order to determine the molecular structures, spectral databases were searched and fragmentation prediction software was used. This approach enabled structural elucidation of novel compounds potentially useful as biomarkers in diagnostics and follow-up of IEM patients. Two new conjugates, glutamyl-glutamyl-phenylalanine and phenylalanine-hexose, were identified in plasma of phenylketonuria patients. These novel markers showed high inter-patient variation and did not correlate to phenylalanine levels, illustrating their potential added value for follow-up. As novel biomarkers for 3-hydroxy-3-methylglutaryl-CoA lyase deficiency, three positional isomers of 3-methylglutaconyl carnitine could be detected in patient plasma. Our results highlight the applicability of current accurate mass multistage fragmentation techniques for structural elucidation of unknown metabolites in human biofluids, offering an unprecedented opportunity to gain further biochemical insights in known inborn errors of metabolism by enabling high confidence identification of novel biomarkers.
- MeSH
- acetyl-CoA-C-acetyltransferasa krev nedostatek MeSH
- biologické markery analýza krev chemie MeSH
- chemická frakcionace metody MeSH
- chromatografie kapalinová MeSH
- fenylketonurie krev diagnóza MeSH
- lidé MeSH
- metabolické nemoci krev diagnóza MeSH
- metabolom MeSH
- metabolomika metody MeSH
- molekulární konformace MeSH
- reprodukovatelnost výsledků MeSH
- software MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vrozené poruchy metabolismu aminokyselin krev diagnóza MeSH
- vrozené poruchy metabolismu krev diagnóza MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Práca predkladá komplex imunologických a imunochemických vyšetrení pacientov s pracovnou diagnózou Meniérova choroba a progresívna senzorineurálně porucha sluchu. Všetky testy boli založené na báze solubilizovaných antigénov ľudskej kochley. Izolované proteíny boli charakterizované SDS PAGE. Pre stanovenie imunologickej podstaty ochorenia sme použili nasledovné testy: imunobloting, ELISA, CD69 analýza periférnych CD3+ lymfocytov prietokovou cytometriou po stimulácii kochleárnymi antigénmi a lymfocyt transformačný test. Testy poukázali na nepravidelnosť tvorby autoprotilátok a na nepravidelnosť aktivizácie periférnyc h cirkulujúcich CD3+ lymfocytov u pacientov, čo môžeme parciálne uzavrieť ako dôsledok lokálneho poškodenia kochley alebo expresie patologických povrchových štruktúr sprístupnenia normálne sa nevyskytujúcich lokálnych antigénov v dôsledku rôznych stimulov (vírusové poškodenie, tlak endolymfy, a pod.). Na základe týchto výsledkov nie je možné vysloviť podporu tvrdeniu, že Meniérova choroba je etiologicky jednoznačne autoimunitné ochorenie. Je však možné vysloviť názor, že v dôsledku navodenia autoimunitného procesu prejavy ochorenia progredujú.
The authors submit a complex of immunological and immunochemical examinations of patients with the working diagnosis of Méniére's disease and progressive sensorineural hearing disorder. All tests were made on the basis of solubilized antigens of the human cochlea. Isolated proteins were characterized by SDS PAGE. For assessment of the immunological basis of the disease the authors used the following tests: immunoblotting, ELISA, CD69 analysis of peripheral CD3+ lymphocytes by flow cytometry after stimulation with cochlear antigens and the lymphocyte transformation test. The tests revealed the irregular formation of autoantibodies and irregular activation of periipheral circulating CD3+ lymphocytes in patients. This can be partially considered the result of local damage of the cochlea or expression of pathological surface structures and availability of normally not present local antigens as a result of various stimuli (viral damage, pressure of endolymph etc.). Based on these results the view can be supported that Méniér e's disease is from the etiological aspect beyond doubt an autoimmune disease. It is however possible that as a result of the induction of the however possible that as a results of the induction of the autoimmune process the manifestations of the disease have a progressive trend.
Twenty known Amaryllidaceae alkaloids of various structural types, and one undescribed alkaloid of narcikachnine-type, named narcieliine (3), have been isolated from fresh bulbs of Zephyranthes citrina. The chemical structures of the isolated alkaloids were elucidated by a combination of MS, HRMS, 1D and 2D NMR, and CD spectroscopic techniques, and by comparison with literature data. The absolute configuration of narcieliine (3) has also been determined. Compounds isolated in a sufficient quantity were evaluated for their in vitro acetylcholinesterase (AChE; E.C. 3.1.1.7), butyrylcholinesterase (BuChE; E.C. 3.1.1.8), and prolyl oligopeptidase (POP; E.C. 3.4.21.26) inhibition activities. Significant human AChE/BuChE (hAChE/hBuChE) inhibitory activity was demonstrated by the newly described alkaloid narcieliine (3), with IC50 values of 18.7 ± 2.3 µM and 1.34 ± 0.31 µM, respectively. This compound is also predicted to cross the blood-brain barrier (BBB) through passive diffusion. The in vitro data were further supported by in silico studies of 3 in the active site of hAChE/hBuChE.
- MeSH
- acetylcholinesterasa chemie metabolismus MeSH
- alkaloidy chemie izolace a purifikace farmakologie terapeutické užití MeSH
- Alzheimerova nemoc farmakoterapie patologie MeSH
- Amaryllidaceae chemie metabolismus MeSH
- butyrylcholinesterasa chemie metabolismus MeSH
- cholinesterasové inhibitory chemie metabolismus farmakologie terapeutické užití MeSH
- hematoencefalická bariéra účinky léků metabolismus MeSH
- katalytická doména MeSH
- kinetika MeSH
- lidé MeSH
- magnetická rezonanční spektroskopie MeSH
- molekulární konformace MeSH
- simulace molekulového dockingu MeSH
- vazebná místa MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
New unknown impurity at m/z 421.15 was observed during the accelerated stability analysis (40 °C/75% relative humidity) in the multi-component tablets of amlodipine besylate by reversed-phase ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS). UHPLC-MS and nuclear magnetic resonance (NMR) techniques were employed to identify and fully characterize the degradation compound. The degradation product was unambiguously identified as 3-ethyl 5-methyl 4-(2-chlorophenyl)-6-methyl-2-(morpholin-2-yl)-1,4-dihydropyridine-3,5-dicarboxylate and mechanism of its formation was proposed. It was confirmed that the degradation product was formed by the reaction of amlodipine with formaldehyde originating from the excipients present in the dosage form.
- MeSH
- amlodipin chemie MeSH
- časové faktory MeSH
- chromatografie s reverzní fází MeSH
- farmaceutická chemie metody MeSH
- fixní kombinace léků MeSH
- formaldehyd chemie MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- hydrochlorthiazid chemie MeSH
- kontaminace léku * MeSH
- magnetická rezonanční spektroskopie MeSH
- molekulární struktura MeSH
- pomocné látky chemie MeSH
- stabilita léku MeSH
- valsartan chemie MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
Affinity capillary electrophoresis (ACE) and quantum mechanical density functional theory (DFT) calculations have been employed for the investigation of noncovalent interactions between hexaarylbenzene-based receptor (R) and ammonium cation NH(4)(+). Firstly, by means of ACE, the binding constant of the NH(4)R(+) complex in methanol was estimated from the dependence of the effective electrophoretic mobility of the receptor R (in advance corrected by our earlier developed procedure to a reference temperature of 25°C) on the concentration of ammonium ion in the background electrolyte using non-linear regression analysis. The logarithmic form of the apparent binding (stability) constant of NH(4)R(+) complex in the methanolic background electrolyte (25 mM Tris, 50 mM chloroacetate, pH(MeOH) 7.8) was evaluated as log K(NH(4)R) = 4.03 ± 0.15. Secondly, the structural characteristics of NH(4)R(+) complex were determined by DFT calculations.
- MeSH
- benzenové deriváty chemie MeSH
- chromatografie afinitní metody MeSH
- elektroforéza kapilární metody MeSH
- kationty chemie MeSH
- kvartérní amoniové sloučeniny chemie MeSH
- methanol MeSH
- molekulární konformace MeSH
- molekulární modely MeSH
- teplota MeSH
- vodíková vazba MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
