DISCUSSION 511 
tion, particularly in some individuals. One person was quite different from another. Some went for 
4 hours with a six-fold elevation of glutamine in plasma and very little change in the erythrocytes. 
At other times there would be a very slight and gradual elevation followed by a fall in erythrocytes. 
The literature suggested some differences in perfusion as compared to ingestion studies. On one 
occasion, after feeding glutamine, we gave 250 ml of saline by slow intravenous drip over a period 
of about 24 hours. This was the only time that we saw plasma and red cells go up and down to- 
gether. We withdrew the needle to stop the saline infusion, and observed during the remainder of 
the study that plasma and red cell glutamine were not at the same level each time as before. This 
study illustrates the ease with which 7v vivo permeability can be changed to resemble the 7 vitro 
situation. 
CHRISTENSEN: The entrance of glycine is very slow, of course, in the red cells. I will speak of 
that this afternoon. I do want to ernie eta that the cellular levels of the free amino acids are ina 
dynamic equilibrium with the extracellular levels. For the liver or the Ehrlich cell and for most 
amino acids, the response is extremely fast; for muscle or the erythrocyte and for such an amino 
acid as glycine the response is considerably slower. But we know of no exception among the tissues 
of higher animals: the cellular level of an amino acid needs to be related to the extracellular level 
and should not be considered in isolation. 
TALLAN: Dr. MussIni mentioned the presence of bound aminoacids in brain. This tiesin with what 
Dr. MITcHELI was discussing yesterday. I think that most of the “bound amino acids” can be accoun- 
ted for by acetylaspartic acid, glutathione, and glutamine. In normal rat brain we find about 6.7 
wmole/g of acetylaspartic acid, which, after hydrolysis, would give 89 mg of aspartic acid per 100 g 
of tissue. There are 1.6 wmole/g of glutathione, which would give rise to 55 mg/1oo g of glycine, 
glutamic acid and cysteine, and 5.4 wmole/g of glutamine, which would yield 79 mg/1oo g of 
glutamic acid. Any glutamine that had cyclized to pyrrolidone carboxylic acid during the prepara- 
tion of the extract would not appear on the chromatogram, but would yield glutamic acid upon 
hydrolysis. If the whole extract is to be analyzed with ninhydrin before and after hydrolysis, the 
expected increase in ninhydrin-reactive compounds arising from the three substances I mentioned, 
calculated as “leucine”, agrees very well with Dr. Mussint1’s figure of about 200 mg per cent of bound 
amino acids. I do not think therefore, that there can be very much peptidic material present. 
L. Mitrer: I just wanted to ask a few minor questions of technique of Dr. TALLAN. You mentioned 
the use of perchloric acid in preparing amino acids as a non-protein filtrate, and I wonder whether 
this affords a special advantage over the Hamilton-Van Slyke picric acid procedure that you did 
not make clear in your presentation. Then, again, Dr. TALLAN gave some quantitative results of 
glutamine content, and I thought, perhaps on the basis of inadequate information, that the con- 
ventional MoorE, SPACKMAN AND STEIN procedure does not give accurate glutamine levels. I 
wondered how these were obtained. 
TALLAN: Perchloric acid is somewhat easier to use than picric acid; removal of the excess with 
KOH is simpler than the removal of excess picric acid with Dowex 2. With brain, perchloric acid 
gives a crystal clear extract, whereas a picric acid extract is usually cloudy and more difficult to 
work with. Our glutamine figures are really minimal values, uncorrected for any possible losses. 
However, the values we get are quite reproducible for a given extract. 
H. RoSENBERG: Dr. ROBERTS mentioned serine ethanolamine phosphodiester in connection with 
the chick embryo. We have looked at this and found that eggs contain none, but as the embryo 
develops this compound appears on the fourteenth day, and can be detected from then on. We 
always examined extracts of whole embryos, without bothering about organ distribution. 
REFERENCES 
1 R. H. McMenamy, C. C. Lunp anp J. L. Oncry, J. Clin. Invest., 36 (1957) 1672. 
ra RG Jel. Mc Menamy, C.C. Lunn, G. J. NEVILLE anp D. F. H. Wattacn, J. Clin. Invest., 39 
(1960) 1675. 
