Determination of rat brain kynurenic acid by column‐switching HPLC with fluorescence detection

Abstract

Kynurenic acid (KYNA), one of the tryptophan metabolites, serves as an endogenous antagonist of N-methyl-d-aspartate and the alpha7 nicotinic receptors in mammalian brains. In the present study, the column-switching high-performance liquid chromatography (HPLC) method we developed for plasma KYNA was extended and validated for the determination of brain KYNA. Rat cerebrum, cerebellum and brainstem homogenates were deproteinized with acetone, and the extracts reconstituted with the mobile phase were injected onto the HPLC. In spite of the facile pretreatment, the fluorescence peak of KYNA in the cerebrum, cerebellum and brainstem was clearly observed with no interfering peaks. Intra- and inter-day precisions [relative standard deviation (%)] and accuracies [relative mean error (%)] were satisfactory (< +/-5.8%). The concentrations of KYNA in rat cerebrum, cerebellum, and brainstem were 224 +/- 65.8, 606 +/- 191, and 323 +/- 114 fmol/mg protein (n = 5), respectively. The proposed HPLC method will be a useful tool for pharmacokinetic and pharmacological researches on brain KYNA.

Changes in Extracellular Kynurenic Acid Concentrations in Rat Prefrontal Cortex After d-Kynurenine Infusion: An In vivo Microdialysis Study

“…Column-switching high-performance liquid chromatography (HPLC) [32][33][34] was performed for the fluorescence determination of KYNA levels in the microdialysis samples by using a previously reported technique [31]. The reported KYNA recovery through the probe, with this technique, is 20.4 ± 2.76% [31].…”

Optimization of Zn2+-containing mobile phase for simultaneous determination of kynurenine, kynurenic acid and tryptophan in human plasma by high performance liquid chromatography

“…Normally ultraviolet (UV) detection was employed to determine KYN [6][7][8][9][10][11] and fluorescence detection was used to measure KYNA and TRP [5][6][7][8][9][10][12][13][14][15][16][17]. Besides, some other techniques were also reported to determine TRP and kynurenines, e.g., measuring KYN and TRP by coulometric detection [18]; KYNA by capillary electrophoresis with laser-induced fluorescence detection [19]; and TRP and related compounds by high performance liquid chromatography-mass spectrometry (HPLC-MS) [20][21][22].…”

Fluorimetric assay for D-amino acid oxidase activity in rat brain homogenate by using D-kynurenine as a substrate

“…Although endogenous KYNA is present in rat brain tissues, the concentration is reported to be relatively low, approximately 200-600 fmol/mg protein among the tissues assayed here (28). In this previous study, a centrifugation fractionation procedure was not carried out, while the assay here uses the pellet fractionby centrifugation.…”