Detection of four different amino acid neurotransmitters in cultured rat neurons and the culture medium by precolumn derivatization high-performance liquid chromatography

Abstract

We validated and used a high-performance liquid chromatography procedure for the determination of four different amino acid neurotransmitters in cultured rat neurons and used culture medium. Samples were derivatized using 2,4-dinitrofluorobenzene and the amino acids were separated on a C18 column. The method yielded good reproducibility and sensitivity for the quantification of the four free amino acid neurotransmitters, with average recovery factors of 80.25-118.43%, an intraday precision of 0.09-0.17%, and an interday precision of 0.62-0.74%. The assay method can be readily utilized as a precise, sensitive, and highly accurate method for the determination of concentrations of the four amino acid neurotransmitters in cultured rat neurons and used culture medium. Using the described methods, we found that the aspartate, glutamic acid, glycine, and γ-aminobutyric acid concentrations (µmol/g protein) in cultured rat neurons were 25.23±0.81, 35.16±0.32, 77.56±4.51, and 62.87±3.12, respectively, whereas their concentrations (µM) in the used culture medium were 18.18±0.82, 24.27±1.01, 107.18±9.56, and 35.78±2.98, respectively.

Reshaping the Binding Pocket of Lysine Hydroxylase for Enhanced Activity

“…Enzyme Activity Assay. The activity of NkLH4 was measured by following the decarboxylation of the cosubstrate oxaloacetate using a succinate test kit (R-Biopharm, Darmstadt, Germany), 39 with L-lysine (99%, Sigma-Aldrich, St. Louis, MO, USA) as the substrate. The assay was performed in 100 μL of 20 mM HEPES buffer (pH 7.5) containing 2 mM DTT, 2 mM αKG, 2 mM L-ascorbic acid and 0.2 mM FeSO 4 • 7H 2 O.…”

“…Reaction mixtures comprising 20 mM HEPES (pH 7.5) with 2 μM purified protein, 5 mM substrate, 2 mM DTT, 2 mM αKG, 2 mM L-ascorbic acid, and 0.15 mM FeSO 4 •7H 2 O were incubated at room temperature for 10 min, and then derivatized with 2,4-dinitrofluorobenzene (DNFB), as previously described. 39 The amino acid derivatives were analyzed using HPLC an XTerraC18 column (5 μm, 4.6 × 250 mm, Waters, Ireland) and Agilent (USA) XWK-multichannel interface at a flow rate of 1 mL/min at 23 °C. The elution of (2S,4R)-4-hydroxylysine was measured using an ultraviolet detector at 360 nm.…”