γ-Aminobutyric acid quantification in small volume biological samples through enzymatically induced electrochemiluminescence.

Abstract

γ-Aminobutyric acid (GABA) is a well-known neurotransmitter that regulates inhibitory neurotransmission in the mammalian central nervous system and participates in several processes outside the brain. A reliable quantification method is needed to determine its role in different physiological and pathological conditions. However, GABA measurements have several challenges because GABA is neither fluorescent nor electroactive, and it is difficult to detect using enzymatic reactions because no oxidases or dehydrogenases have been identified. Several methods have been developed to quantify GABA concentrations based on the instrumentation available, the sensitivity required, and the volume of samples analyzed. Most of these methods use high-performance liquid chromatography (HPLC). Here, we describe a method for quantifying GABA concentrations in small volume samples using enzymatically-induced electrochemiluminescence with the well-known GABAse complex, which produces glutamate for use in a luminescent reaction with glutamate oxidase and luminol in an electrochemiluminescence cell. The luminescence obtained was proportional to the GABA concentrations in the micromolar range (1-1000), with linear r2 values > 0.95. GABA standards were treated with the enzymatic reactors to generate glutamate (Glu), which was measured simultaneously with an HPLC technique, to validate this new procedure. The assay was further used to determine GABA concentrations in hippocampal extracts. This alternative may be used to quantify GABA levels in fluid samples, such as microdialysates, other perfusates and tissue extracts. Thus, the method presented here is a good alternative for monitoring GABA levels with good sensitivity compared with the traditional methods that are still in use.