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-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 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.


RESULTS
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.


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]beta-alanine indicate that GABA and beta-alanine transaminases are identical.
Metabolic Unit, Department of Clinical Chemistry, University Hospital Vrije Universiteit, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.
D S
DS
Schor
E A
EA
Struys
B M
BM
Hogema
K M
KM
Gibson
C
C
Jakobs
D000612:4-Aminobutyrate Transaminase / Q000737:chemistry / Q000378:metabolism*; D000410:Alanine Transaminase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007201:Indicator Dilution Techniques; D008214:Lymphocytes / Q000201:enzymology*; D009587:Nitrogen Isotopes; D021241:Spectrometry, Mass, Electrospray Ionization; D015091:beta-Alanine / Q000378:metabolism
D009587:Nitrogen Isotopes; D015091:beta-Alanine; D000612:4-Aminobutyrate Transaminase; D000410:Alanine Transaminase