Development of a stable-isotope dilution assay for gamma-aminobutyric acid(GABA) transaminase in isolated leukocytes and evidence that GABA and beta-alanine transaminases are identical

Background: Several methods have been published for measuring gamma -aminob utyric acid transaminase (GABA-T) activity, but these methods are either im practicable because of the use of radioisotopes or insufficientfy sensitive to determine small enzyme activities in leukocyte extracts. We developed a direct and sensitive enzyme method. Methods: We developed a stable-isotope dilution method for the measurement of [N-15]glutamic acid derived from [N-15]GABA and alpha -ketoglutaric acid , catalyzed by GABA-T. The method for analysis of [N-15]glutamic acid compr ised a solid-phase extraction procedure to isolate this analyte from incuba tion samples. After derivatization, [N-15]glutamic acid was quantified by g as chromatography-mass spectrometry relative to its H-2(5)- labeled interna l standard. In addition to [N-15]GABA, [15N]beta -alanine was a cosubstrate . Results: GABA-T-deficient lymphoblasts showed diminished enzyme activity, w ith both [N-15]GABA and [N-15]beta -alanine as substrate. Vigabatrin inhibi ted the enzyme activity for both substrates. Conclusions: The activity of GABA-T can be accurately determined by our pro cedure using N-15-labeled substrate, measuring the formation of [15N]glutam ic acid. Our results with [15N]beta -alanine indicate that GABA and beta -a lanine transaminases are identical. (C) 2001 American Association for Clini cal Chemistry.