Further studies on brain gamma-aminobutyric acid metabolism after administration of pyridoxine to vitamin B-6-deficient suckling rats.

Convulsions due to vitamin B-6 deficiency in humans can be controlled by the administration of pyridoxine, and the symptoms subside rapidly (Coursin, 1954). If derangement of y-aminobutyric acid metabolism were responsible for these convulsions, it would be expected that a return to normal y-aminobutyric acid metabolism occurs very rapidly after the pyridoxine administration. In vitamin B-&deficient suckling rats (Bayoumi & Smith, 1972), the amount of glutamate decarboxylase activity was shown to be decreased to 40% of control values. After intraperitoneal administration of pyridoxine hydrochloride (lOmg), the activity recovered almost immediately. Within 2min the activity was four times that in the untreated deficient animals (Bayoumi et al., 1974). The concentration of y-aminobutyric acid would also be expected to rise, following the same pattern as the glutamate decarboxylase. However, a lag of 20min occurred before the y-aminobutyric acid concentration began to rise. In order to resolve this apparent discrepancy, vitamin B-6-deficient suckling rats were given pyridoxine hydrochloride (10mg) by intraperitoneal injection at timeintervals ranging from 1 to 60min before they were killed. The animals were also given an intraventricular injection of ~-[U-'~C]glutamic acid (0.14pmol), 15min before they were killed. The brains were then prepared as for amino acid (Bayoumi et al., 1974). The y-amin~['~C]butyric acid and total brain glutamate concentrations of the preparations were determined. The y-aminobutyric acid was separated by a two-dimensional process by using, first, paper chromatography (butan-1-ol-acetic acid-water, 12: 3 : 5, by vol.) on Whatman no. 1 paper, followed by high-voltage electrophoresis at 3 kV, 60mA at 90" to the direction of chromatography, in pyridine-acetic acid buffer, pH5.3. The radioactive spots corresponding to y-aminobutyric acid were located with a Pullan spark-chamber radiochromatogram scanner, cut out and counted for radioactivity in toluene scintillator [0.3 % 2,5-diphenyloxazole, 0.03 % 1,4-bis(5-phenyloxazol-2-yl)benzene] in a Packard 3385 Tri-Carb liquid-scintillation counter. The glutamic acid was measured after one-dimensional paper electrophoresis under the same conditions as above. The spots were quantitatively stained with ninhydrin (1 % in acetone). After heating, the glutamic acid spots were cut out and eluted in 5ml of 80% (v/v) ethanol. In order to compare relative glutamate decarboxylase activities in vivo, calculations are made assuming that, since the injected [14C]glutamic acid is negligible in amount compared with the total endogenous glutamic acid, the proportion of endogenous glutamate converted into y-aminobutyric acid approximates to the proportion of injected ['4C]glutamic acid converted into y-amin~['~C]butyric acid. Hence, the glutamate decarboxylase activity in vivo is expressed here as y-aminobutyric acid (pmol) produced/h per brain. This is found from