Schor D S, Struys E A, Hogema B M, Gibson K M, Jakobs C
Metabolic Unit, Department of Clinical Chemistry, University Hospital Vrije Universiteit, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.
Clin Chem. 2001 Mar;47(3):525-31.
Several methods have been published for measuring gamma-aminobutyric acid transaminase (GABA-T) activity, but these methods are either impracticable because of the use of radioisotopes or insufficiently sensitive to determine small enzyme activities in leukocyte extracts. We developed a direct and sensitive enzyme method.
We developed a stable-isotope dilution method for the measurement of [15N]glutamic acid derived from [15N]GABA and alpha-ketoglutaric acid, catalyzed by GABA-T. The method for analysis of [15N]glutamic acid comprised a solid-phase extraction procedure to isolate this analyte from incubation samples. After derivatization, [15N]glutamic acid was quantified by gas chromatography-mass spectrometry relative to its 2H5-labeled internal standard. In addition to [15N]GABA, [15N]beta-alanine was a cosubstrate.
GABA-T-deficient lymphoblasts showed diminished enzyme activity, with both [15N]GABA and [15N]beta-alanine as substrate. Vigabatrin inhibited the enzyme activity for both substrates.
The activity of GABA-T can be accurately determined by our procedure using 15N-labeled substrate, measuring the formation of [15N]glutamic acid. Our results with [15N]beta-alanine indicate that GABA and beta-alanine transaminases are identical.
