NET PRODUCTION OF γ‐AMINOBUTYRIC ACID IN ASTROCYTES IN PRIMARY CULTURES DETERMINED BY A SENSITIVE MASS SPECTROMETRIC METHOD

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Abstract
—: A sensitive and specific high resolution mass spectrometric method, using a deuterium labelled internal standard, was developed for the quantitation of GABA in biological materials. GABA was converted to the DNS‐γ‐butyrolactam, isolated by thin layer chromatography and quantitated by the integrated ion current procedure. The method was used to measure the GABA content of cultured astrocytes (0.0545 nmol/mg protein, depending upon the culturing conditions), of fresh media (0.02–0.04 μM) and of conditioned media (after culturing in the absence of added GABA: 0.02–1.8 μM, depending upon the culturing conditions). An estimate of the rate of GABA production was obtained from the concentration in conditioned media and in the cells when GABA degradation was inhibited by AOAA (13 μM). The production of GABA was negligible in the cells grown under ordinary conditions but increased to 0.3 nmol/h per mg protein in cells which had been grown in the presence of 0.25 mM‐ dibutyryl cyclic AMP for 1 week. This value is of the same order of magnitude as the GAD activity observed in extraneurónal tissue. However, the GABA production measured mass spectrometrically was much lower than that of labelled GABA from [U‐14C]glutamate. The latter value was non‐reproducible and varied from one batch of radioactive glutamate to another.