Details

Autor(en) / Beteiligte

• Katalin, Cseh Edina

Titel

HPLC Method Development for the Assessment of Tryptophan Metabolism and its Application in a Complete Freund’s Adjuvant Model of Orofacial Pain

Ort / Verlag

ProQuest Dissertations & Theses

Erscheinungsjahr

2020

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• The pathomechanism of orofacial pain and headache disorders, including migraine is complex and need further elucidation. Nevertheless, the activation and sensitization of the trigeminovascular system has especially important role in the development of symptoms. For better understanding of the pathomechanism of these disorders, animal models with the activation of nociceptive pathways of the trigeminovascular system are used. Complete Freund’s adjuvant (CFA)-induced orofacial pain model serves for the induction of peripheral inflammation. Therefore, the investigation of the neurochemical profile of this model could provide meaningful information regarding pain processing.In the field of neuroscience, the information obtained via the measurement of biomarkers may aid the diagnosis, prevention and treatment of different neurological disorders. The main purpose of the bioanalytical assessments is to reveal whether there are changes on molecular levels during the development, course and treatment of a disease, either from a clinical or preclinical point of view.Our aim was to investigate the concentration changes of some biomarkers, including glutamate (Glu), γ-aminobutyric acid (GABA), and serotonin, and in light of its influence on glutamatergic neurotransmission, we further expanded the investigation of the neurochemical profile with the measurement of the level of kynurenic acid (KYNA) and its precursors in the kynurenine (KYN) pathway (KP) of tryptophan metabolism. Furthermore, due to the importance of the KP, we also aimed at the method optimization and validation on six different biological matrices, including human plasma and cerebrospinal fluid (CSF), mouse brain and plasma, and rat central nervous system (CNS) and plasma.The CFA model consisted of the CFA (1 mg/ml, 50 μl/animal) injection into the right whisker pad of male Sprague-Dawley rats. The samples were collected 24 and 48 h after injection, whereas the control group rats, injected with saline, were processed at 24 h after injection. Two important brain regions were chosen to determine the concentration of the above-mentioned metabolites in the trigeminal nucleus caudalis (TNC) and somatosensory cortex (ssCX), as both have an important role in the pain processing. We applied high-performance liquid chromatography (HPLC) coupled with UV and fluorescence detection, using separate internal standards for each detector: 3-nitro-L-tyrosine and the newly utilized 4-hydroxyquinazoline-2- carboxylic, respectively, to determine the concentration changes of the metabolites. For further method validation process, carried out on the different biological matrices, the same HPLC method was used in each case, with slight modifications.In the CFA model, our results demonstrated that 24 h after CFA treatment, the level of Glu, KYNA and that of its precursor, KYN, were still elevated in the TNC, all diminishing by 48 h. In the ssCX, significant concentration increases of KYNA and serotonin were found. Regarding the assessment of some TRP metabolites, the method was successfully utilized for measurements from human plasma and CSF, mouse brain and plasma, and rat CNS and plasma. Regarding murine CNS samples, serotonin was successfully measured as well in one single run with TRP, KYN and KYNA. During the method validation, good intra- and inter-day precision values were obtained with coefficient of variation <5%, and bias <6.5% (except the serotonin levels in murine CNS samples), respectively. The recoveries varied between 79.6% and 116%, with all results being in line with the official guidelines.