240 P. SOUPART 
red cell sediment resuspensed in saline and also centrifuged twice. Values obtained 
from the last sediment, after lysis and picric acid deproteinization of the lysate, are 
shown in Table VII under the heading non-corrected for diffusion. Concentrations listed 
in this column suggest that most of the amino acids contained by red cells have 
been lost by diffusion during the preparative steps. This has been controlled in the 
following experiment: a sample has been collected after each step involving eryth- 
rocytes, in order to assess the extent and moment of loss by diffusion. The results 
of these experiments are shown in Table IX, and the following facts can be em- 
phasized: 
1) In erythrocytes which have not been washed, free amino acids may be divided 
in three groups according to their respective concentration. In the first group there 
are 13 free amino acids; their concentration is similar to that observed in the plasma 
(7.e. amino acids having an erythrocytes/plasma concentration ratio close to unity, 
comprised between 0.77 and 1.25). It includes taurine, threonine, serine, alanine, 
a-aminobutyric acid, valine, methionine, isoleucine, leucine, tyrosine, phenyl- 
alanine, and histidine. Such is also the case for urea. The second group includes 
seven free amino acids or amino acid derivatives, which have an intracellular con- 
centration significantly higher than that found in plasma, namely: aspartic acid, 
phosphoserine, glutamic acid, glutamine (and asparagine), ornithine, methionine 
sulfoxides and glycine. Apparently, such is also the case for ammonia. In the third 
group are found three amino acids having an intracellular concentration signifi- 
cantly lower than that found in the plasma, namely: citrulline, arginine and proline. 
One must be cautious when including proline in this group since the presence of 
glutathione in its elution range renders its determination difficult, unless glutathione 
is converted into glutathione S-sulfonate which will be eluted with the front of the 
eluent, but this has not been done in this particular experiment. It is possible there- 
fore that proline has to be included in the first rather than in the third group. The 
finding of a low arginine concentration associated to a high ornithine concentration 
is not surprising since erythrocytes are known to contain arginase®?. 
2) After the three-stage process for erythrocytes spontaneous sedimentation in 
buffered isotonic solution containing 2° polyvinylpyrollidone, it can be seen that 
some of the amino acids are easily lost by diffusion (threonine, proline, valine, 
isoleucine, leucine, tyrosine, phenylalanine, ornithine, lysine and histidine; and 
also urea), while others diffuse rather slowly into the amino acid-free washing 
solution (phosphoserine, taurine, aspartic acid, serine, glutamine (and asparagine), 
glutamic acid, glycine, alanine, cystathionine, B-alanine and 1-methylhistidine; and 
also ammonia). Arginine has completely disappeared presumably as a result of 
continued arginase activity. 
3) The two washings in saline solution introduce a new factor since the solution 
used, although isotonic, is not buffered. After each of these two steps, the intra- 
cellular concentration of taurine, aspartic acid, glutamine (and asparagine), glutamic 
acid, glycine, alanine and also of ammonia, is not impressively lowered, whilst other 
amino acid concentrations continue to drop progressively or even abruptly, which 
drop might presumably be accounted for by the fact that pH conditions are modified 
and that volume contraction might have been induced. 
From such an observation many questions arise. Why is an amino acid such as 
serine apparently so firmly retained in the red cell when threonine leaks away so 
References p. 261/262 
