510 Chairman: G. ROUSER 
CHRISTENSEN : [haveonecomment I would like toaddress to Dr. Soupart. I notice that you correct 
your leucine level for that lost by washing to reach the conclusion that it may be three times as 
great in the red blood cell as in the plasma. When we add radioactive leucine to fresh heparinized 
blood we find that it enters extremely rapidly. In our hands, even with long delays, the ratio fails 
to surpass a value of 1.1, calculated on a water basis. Now, because of the extreme speed with 
which the amino acids with large hydrophobic side chains do move into the red cell, the correction 
for washing must be extremely difficult, and I wonder if, in this case, the correction may have been 
too large. 
Also, I should like to comment to Dr. RoBErts that we interpret quite differently the ease with 
which the levels of the free amino acids of the tissues can be changed. We find that it is desirable to 
consider the tissue levels always with reference to the extracellular level, since we have never found 
a situation where they are not highly responsive to their environmental levels. Suppose you had a 
section of heart muscle that is cut off by infarction. The lack of irrigation of that area could cause 
a backing up of amino acids being lost from the fiber into the interstitial fluid, which would per- 
haps prevent further loss from that area. 
I think, however, a more important source of the disagreement in our philosophies about this 
arises from the low precision that one gets in comparing the amino acid patterns under a given 
change in condition by gazing at a pair of chromatograms. For example, partial hepatectomy 
causes an analytically measured increase of 70 per cent in 24 hours, but has apparently led to a 
rather insignificantly changed chromatogram. I wonder if changes highly significant to metabolic 
processes are not being overlooked in the inspection of chromatograms. 
E Roperts: Using paper-chromatographic methods we have examined the extracellular phase in 
some instances, for example, by studying the ascitic fluid in which ascites tumor cells exist. In 
this case, we found that both cells and fluid showed a remarkable resistance to change. Here there 
is no problem of circulation at all. In some experiments the cells looked like they were virtually 
falling apart, and yet the easily extractable amino acids were retained and did not leak into the 
fluid. There were some changes, but actually, if anything, the easily extractable intracellular pool 
was higher than in the untreated tumors. 
In the case of the heart, we know that big molecules must be leaking out rapidly after infarction 
because there is a remarkable rise in lactic acid dehydrogenase activity and other enzymes in 
blood. I believe this has been traced to an actual leakage of the enzymes from the infarcted heart 
muscle. Thus, this is evidence that there is some interchange taking place between infarcted heart 
muscle and blood. If “backing up” occurred and the interchange were stopped we would get 
autolytic changes, which we know can take place under sterile conditions, as was shown in some 
of the experiments we reported here. We would have expected then an increase in many of the 
detectable constituents. Instead, there was a retention of the initial pattern for quite some time, 
8 hours or so, and then a very slow loss. The kinetics of these changes, I am sure, are quite different 
from the kinetics of other processes, such as loss of large molecules and of the appearance of various 
pathological changes. 
Probably, the most important metabolic aspects of the easily extractable ninhydrin-reactive 
constituents relate to the turnover rates of these constituents in the various tissue compartments. 
There is no question that adrenalectomy, hypophysectomy, and other experimental procedures 
employed produced changes in turnover and possibly in the compartmentalization within the 
tissues studied, but the total concentrations of the various constituents were observed to be 
remarkably constant. It is quite possible, for example, that the total amount of y-aminobutyric 
acid found in a particular brain area is much less important than the quantity that is bound to 
some neuronal structure at which it affects the ionic movements. 
I fully agree that what we have reported today is a very broad descriptive study, the purpose 
of which was to delineate areas for further investigation fully aware of all of the reservations. 
We were greatly surprised, indeed, by the remarkable stability of steady state concentrations of 
the easily extractable ninhydrin-reactive constituents that we detected in our chromatograms. 
RouseErR: Actually, if we look at the studies that Dr. RoBErRtTs reported, some distinct differences 
on the chromatograms can be seen. I know he has been most impressed that an over-all uniformity 
exists, yet there are differences. I think Dr. CHRISTENSEN is pointing out that in contrast he can meas- 
ure various changes. This sort of thing can also be seen on a chromatogram, but it does depend on 
what you are looking at. Dr. RoBERTS’ over-all point is that such changes were not as impressive as 
one might have expected. 
I would like to add some comment about the observations on the red cells. Red cell permeability 
is an intriguing subject, and we began to do some in vitvo control studies in our work to determine 
the extent of change from in vivo conditions. The permeability for amino acids was always differ- 
ent in vitvo judging from the responses seen when we fed glutamine to a person and followed the 
blood level afterwards. When we added so-called physiological saline or a buffer in vitro, the red 
cell became rather freely permeable. It did not concentrate amino acids particularly, but they 
would move in. On the other hand, in in vivo studies the striking thing was the slowness of penetra- 
References p. 511 
