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γ-Aminobutyric acid, glutamate, glycine and taurine analysis using reversed-phase high-performance liquid chromatography and ultraviolet detection of dansyl chloride derivatives

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    As enzyme purification is a labor-intensive, time-consuming, and, therefore, expensive process, this assay was adapted for crude mouse or rat brain homogenates to allow a faster screening of potential GAD inhibitors/activators. DNS-Cl, which has found widespread use in the analysis of amino acids including GABA at very low levels (Cann-Moisan et al., 1990; Kang et al., 2006; Saller and Czupryna, 1989) due to its efficiency, good stability, and low cost, was chosen as the derivatization agent. Another advantage of this derivatization reagent is that it enables detection either by UV absorbance or fluorescence.

  • Simultaneous HPLC determination of gamma amino butyric acid (GABA) and lysine in selected Pakistani rice varieties by pre-column derivatization with 2-Hydroxynaphthaldehyde

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    GABA and other amino acids such as lysine have been determined by LC followed by UV, fluorescence, or colorimetric detection (Clark et al., 2007). Many methods have utilized the pre-column derivatization with orthophthalaldehyde (OPA) (Kehr, 1998; Saller and Czupryna, 1989; Yamamoto et al., 1985; Nasholm et al., 1987; Sawai et al., 2001), phthaldehyde-2-mercaptoethanol (Lee et al., 2010), dansyle chloride (Mazur et al., 2011), 2,4,6-trinitro benzene sulphonic acid (Clark et al., 2007) and 9-fluorenyl-methyl chloroformate (Roohinejad et al., 2009) for the detection of GABA. In this study, 2-hydroxynaphthaldehyde is used as a derivatizing reagent.

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    The aliquot destined for arsenic analysis was terminated as given above. The other fraction used for analyzing glycine, the product of GSH arsenolysis, was subjected to derivatization with dansylchloride immediately after the incubation according to [21] with slight modifications. Briefly, to 30 μl of incubates, 150 μl 5 mM dansylchloride (dissolved in acetone) was added.

  • Protein restriction during early life in rats alters pancreatic GABA<inf>A</inf> receptor subunit expression and glucagon secretion in adulthood

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    Between 200 and 500 islets were isolated from each separate animal for each experimental group, washed twice in Hank’s balanced salt solution (HBSS) (Sigma-Aldrich) and added to 200 to 400 μL of ice-cold extraction buffer (5% formic acid, 1% trifluoroacetic acid [TFA], 1% sodium chloride, 1N hydrochloric acid), dispersed by sonication and stored at −80°C. Proteins were extracted by Sep-Pak C18 reverse-phase chromatography as previously described (34), and insulin and glucagon content were measured by radioimmunoassay (RIA). Rat insulin and glucagon RIA kits (Millipore, Billerica, MA) were used as per manufacturer’s instructions.

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