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Development of a Stable-Isotope Dilution Assay for -Aminobutyric Acid (GABA) Transaminase in Isolated Leukocytes and Evidence That GABA and ß-Alanine Transaminases Are Identical

¹ Metabolic Unit, Department of Clinical Chemistry, University Hospital Vrije Universiteit, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.

² Department of Molecular and Medical Genetics, Biochemical Genetics Laboratory, Oregon Health Sciences University, Portland, OR 97201.
^a Author for correspondence. Fax 31-20-4440305; e-mail C.Jakobs{at}azvu.nl.

Background: Several methods have been published for measuring -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.

Methods: We developed a stable-isotope dilution method for the measurement of [¹⁵N]glutamic acid derived from [¹⁵N]GABA and -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]ß-alanine was a cosubstrate.

Results: GABA-T-deficient lymphoblasts showed diminished enzyme activity, with both [¹⁵N]GABA and [¹⁵N]ß-alanine as substrate. Vigabatrin inhibited the enzyme activity for both substrates.

Conclusions: 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]ß-alanine indicate that GABA and ß-alanine transaminases are identical.

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