An HPLC-based assay for improved measurement of glutamate decarboxylase inhibition/activation

• Published in: Neurochemistry international Vol. 161; p. 105433
• Main Authors: Genčić, Marija S., Stojanović, Nikola M., Mladenović, Marko Z., Radulović, Niko S.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.12.2022
• Subjects: Animals; Chromatography, High Pressure Liquid; Crude mice and rat brain homogenates; gamma-Aminobutyric Acid; Glutamate Decarboxylase; Glutamic Acid; In vitro screening assay; Mice; Rats; Reproducibility of Results; γ-aminobutyric acid; In vitro screening assay; Crude mice and rat brain homogenates; Glutamate decarboxylase; γ-aminobutyric acid
• DOI: 10.1016/j.neuint.2022.105433
• ISSN: 0197-0186

L-Glutamic acid decarboxylase (GAD) is an enzyme that ensures the balance between the levels of two neurotransmitters, γ-aminobutyric acid (GABA) and L-glutamic acid (L-Glu), necessary for proper brain functioning. A reduction in the concentrations of GABA and/or GAD activity has been implicated in the symptoms associated with epilepsy, which could be plausibly alleviated by the application of GAD activators. As any unnecessary interference in GAD catalytic activity could be detrimental, it is important to study whether CNS (or other) drug candidates act on GAD or not. The ability to identify and reduce this risk early could significantly improve the process of drug development. Although many methods for measuring GAD activity in various biological samples have been described, only few (such as manometric and radiometric) were adopted as in vitro assays for the screening of potential GAD inhibitors/activators. However, these methods require specialized equipment and/or an expensive radiolabeled substrate, and may have sensitivity and/or reliability issues. Therefore, this study aimed to develop an HPLC-DAD-based assay that would allow a simple and more accurate measurement of GAD inhibition or activation using unpurified mice or rat brain homogenates. This assay is based on the quantification of GABA, formed during the enzymatic reaction, after its derivatization with dansyl chloride. Various parameters were evaluated to optimize the assay procedure (e.g. homogenate volume, incubation time, DMSO content, GAD, GABA, and dansyl-GABA stabilities). This assay was validated for pharmacological screenings using 3-mercaptopropionic acid and gallic acid and GAD obtained from different experimental animals. [Display omitted] •Glutamate decarboxylase (GAD) regulates the interconversion of L-Glu to GABA.•Only 2 methods with serious issues are in use for in vitro measurements of GAD.•An improved simple HPLC-based assay for GAD activity was developed.•No need for specialized equipment, expensive reagents, and pure enzyme use.•Possibility of sample storage and different enzyme sources utilization.