500 C. F. BAXTER AND E. ROBERTS 
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Figs. 5-10. Figs. 5,6: Effect of thiosemicarbazide upon the concentration of amino acids of brain 
cortex of rats. Injected dose: 20 mg/kg body wt. Figs. 7-10: Irreversibility of thiosemicarbazide 
effect with anticonvulsant doses of pyridoxine. Injected dose: 400 mg/kg body wt. Protocol of 
experiment: Fig. 5, control animals. Fig. 6; effect of thiosemicarbazide injected intraperitoneally. 
Some animals were decapitated 80 min after injection (Fig. 6); other animals were given anti- 
convulsant doses of pyridoxine at 80 min after thiosemicarbazide injection. These animals were 
decapitated 15, 45, 65, and 85 min after the vitamin B, administration (Fig. 7-10). This series 
shows that thiosemicarbazide depressed primarily levels of GABA (see arrow, Figs. 5, 6) and 
that subsequent administration of pyridoxine did not reverse this effect within 85 min. Each 
chromatogram represents the free or loosely bound amino acids of 25 mg of brain tissue (wet wt.) 
Footnote to Figs 1-4. 
* All chromatograms shown in this paper were developed at 26° by descending two-dimensional 
chromatography. Point of origin is at the lower right-hand corner of each chromatogram. The 
first solvent (horizontal, right to left) was water-saturated phenol for 18 h (a few drops ammo- 
nium were added to the chromatography boxes). The second solvent (vertical, bottom to top) 
was water-saturated lutidine for 24 h. These systems have been used and described previously** 
Clear tissue extracts were prepared from ethanol (75%) homogenates by high speed centrifugation. 
Extracts were evaporated to dryness and resuspended in a suitable amount of water. No more 
than 25 wl was applied to the paper at one time and no more than 50 wl per spot. Unless stated 
to the contrary, each chromatogram represents the free or loosely bound amino acids of 30 mg 
of tissue (wet wt.). Amino acids were visualized by spraying with ninhydrin in methanol (3 g 
ninhydrin dissolved in 5 lb of methanol). Photographs were taken no earlier than 12h and no 
later than 48 h after applying the spray reagent. 
In Fig. 1 the numbered spots correspond to the following amino acids: (1) y-aminobutyric acid ; 
(2) alanine (and below it glutamine); (3) ethanolamine phosphate (and above it glutamic acid; 
the spot to the right of glutamic acid is aspartic acid); (4) taurine; (5) glycine (the upper right- 
hand portion of this spot is serine). The compound m, a marker is a-amino-n-butyric acid. 
References p. 508 
