ROUND TABLE DISCUSSION uy 
for the induction of seizures by thiosemicarbazide. When chemicals which are not ordinarily 
present are applied to living cells, one must keep in mind the myriad possible ways in which 
some of them may act and not merely focus on the one or two enzyme systems which are under 
investigation. Both hydroxylamine and thiosemicarbazide may form derivatives with a large 
variety of naturally-occurring carbonyl compounds, which derivatives may have various actions 
of their own. Thiosemicarbazide can chelate zinc. Zinc is required for the activity of various 
dehydrogenases and carbonic anhydrase. The hippocampus, an area of brain that is particularly 
sensitive to seizures, has a high zinc content. Could at least part of the effect of thiosemicarbazide 
be related to its zinc-chelating capacity? 
There is now evidence that yABA may be bound physically to some constituent found in the 
tissue of the central nervous system!’. It may well be that it is not the total quantity of yABA, 
but the amount of bound yABA which will determine the effects of this substance on the con- 
ductance of potassium and/or chloride ions, which is presently believed to be the way in which 
yABA influences neuronal activity. An agent which can either inhibit or enhance binding of 
yABA may have an effect on seizure threshold and other neuronal properties regardless of the 
effect on total content of yABA in a particular area of brain. 
Thus, as in almost every other field of study related to the nervous system, the area related to 
yABA has turned out to be more complicated than one would have anticipated or wished. 
TALLAN: Dr. RoBerts has so well summarized the current status of studies on the amino acids in 
brain that there is very little one could add. I would like, though, to bring up one technical 
question for discussion. That is, what is the best way to extract the free amino acids of brain? 
Dr. Musstntuses picricacid, Dr. Roperts and his group use ethanol, and I have been using perchloric 
acid. There have been a number of side discussions on this point during the past few days, con- 
cerned with whether these are really comparable. For example, I found earlier that the use of 
TCA, followed by ether to remove the excess, could result in a loss of tyrosine. Dr. MAyRON has 
asked me whether perchloric acid might not leave glycoproteins in solution, as is the case with 
blood serum. Dr. BAXTER has brought up the question of whether perchloric acid extracts as much 
yABA from the brain as does ethanol. These are points we are going to check. Has anyone else 
had experiences along this line? 
E. Roserts: We have not done the refined quantitative work, in general, outside of determina- 
tions of y-aminobutyric acid and glutamate, which you have performed on your columns, Dr. TALLAN. 
Early in the game we tried various procedures, such as heat coagulation followed by dialysis, 
trichloroacetic acid precipitation and extraction of the trichloroacetic acid with ether, preparation 
of perchloric acid extracts and removal of excess perchlorate as the potassium salt. We ran chro- 
matograms and got virtually identical chromatograms on extracts prepared by these various 
methods. When yABA is elevated after treatment with hydroxylamine, the effect can be seen by 
all the methods. Routinely we have been preparing alcoholic extracts, which are evaporated to 
dryness and taken up in water saturated with picric acid. I do not believe that it is a critical 
matter which of the above methods is used for study of free amino acids. When one studies 
substances which are liberated upon acid hydrolysis, the choice of method will be critical since 
what is precipitated and what is left in solution depends on conditions. 
KiHara: I would like to ask Dr. RoBerts if he has considered the possibility that when hydroxyl- 
amine does raise yABA it might act by inhibiting pyridoxal phosphate formation. 
Roserts: I think that thiosemicarbazide lowers yABA because it inhibits pyridoxal kinase. 
The results which Dr. BAXTER reported at this Symposium indicate very clearly that the yABA 
transaminase is inhibited very remarkably by hydroxylamine while the glutamic decarboxylase is 
not. Our idea at present is that hydroxylamine acts by inhibiting the yABA transaminase prefer- 
entially to the glutamic decarboxylase because it forms a stable oxime derivative of the pyridoxal 
phosphate which remains attached to the apoenzyme, and that the oxime may be hydrolyzed 
slowly or the inactive coenzyme may take a relatively long time to dissociate from the apo- 
enzyme. Once free, the apoenzyme can reassociate with active coenzyme present in the tissues. 
L. Mixter: In speaking about actions of drugs on the concentration of one or another substance 
in the brain, I believe one ought to try at least to designate what tissue is being studied; that is 
to say, whether it was the cerebral cortex or whether one has really looked carefully at the mid- 
brain and medulla, where very powerful effects can be mediated. In just removing cerebral cortex, 
as one is more apt to do in an undiscriminating brain study, you may totally miss a point. So 
I wonder, for example, in your case, Dr. WitTeR did brain analysis clearly include the medulla and 
midbrain ? 
Witter: These were included in this study. 
H. RosENBERG: What were the concentrations of dieldrin which you found produced the near- 
lethal and lethal effects? Just for information purposes, if you happen to have it, how would 
this concentration compare with the amounts of dieldrin generally applied over areas of agri- 
cultural land and what would be a lethal dose for sheep? 
Witter: The first part of your question was answered in my talk. We gave the animals 160 mg/ 
References p. 524 
