FREE AMINO ACIDS IN BLOOD. I 359 
In vitro incubation studies with blood from chronic lymphatic leukenna patients 
In vitro incubations were performed to determine what arteifacts might arise during 
the processing of blood samples. Incubation of whole blood from untreated patients 
with chronic lymphatic leukemia disclosed that marked changes in the amino acid 
composition of plasma, leukocytes, and erythrocytes took place at 37°. The complete 
degradation of glutamine in cells and plasma was observed, and a marked increase 
of glutamic acid and alanine was observed in both plasma and cells. A similar study 
of whole blood from a patient with a high leukocyte count with 0.7 mg/ml of glutamine 
added followed by incubation at 37° with shaking at 120 cycles/min demonstrated 
that glutamine was completely metabolized in a little over 4 h. Incubations of white 
cells in plasma and red cells in plasma were then carried out to determine the relative 
metabolic activity of the leukocytes and erythrocytes. 15 7m vitvo incubations in all 
were carried out with chronic lymphatic and chronic granulocytic leukemia blood 
samples. 
One study with added glutamine was carried out as follows. 50 ml of blood was 
drawn into heparin from a patient with chronic lymphatic leukemia and cells and 
plasma separated as described above. Glutamine (0.25 mg/ml) was added to plasma. 
18 ml of plasma was then mixed with 18 ml of erythrocytes, and 17 ml of the plasma 
was mixed with 2.7 ml of packed leukocytes (1300 « g). The incubations were carried 
out ina Warburg bath at 37° with shaking at 120 cycles/min in siliconed Erlenmeyer 
flasks in an atmosphere of air. Samples were withdrawn at the following times after 
mixing: 5, 10, 30, 45, 60, 90, 120, 200, and 240 min. An aliquot of 1.5—2.0 ml of the 
plasma plus leukocyte mixture was removed each time, and 3.0-3.3 ml of the red 
cell plus plasma mixture was removed for each sample. The samples were spun and 
the volume of plasma and packed cells was noted in each case. Approximately 5 min 
was required for satisfactory separation of cells and plasma at 1300 x g. The plasma 
was aspirated as completely as possible and the cells were treated (without washing) 
with alcohol for extraction. The last trace of plasma above the packed cells was 
removed with a small swab. 
During the course of the erythrocyte incubation a trace of hemolysis was evident 
first at 160 min, but was still very slight at 240 min. The pH of the plasma at the 
beginning of the erythrocyte incubation was 7.5 and rose to 8.1 at the end of the 
incubation. The pH of plasma rose from 7.5—8.3 in the leukocyte incubation flask. 
At the end of the erythrocyte incubation the erythrocyte volume had increased 
from an initial value of 45°% to 47% (volumes determined at 1300 x g for 5 
min). The initial packed volume of leukocytes (1300 x g, 5 min) was 12%. The 
cells swelled gradually to a value of 14.5°% at the end of the incubation (21% 
increase). 
Figs. 13-16 show the plasma samples and Figs. 17-20 the leukocyte samples 
obtained at the beginning of the incubation and after 60, go, and 240 min. The 
complete absence of glutamine from the white cells was first evident at 60 min 
(Fig. 18) and the complete disappearance from plasma was evident at go min (Fig. 15). 
White cells lost taurine to the plasma and large increases of alanine and glutamic 
acid with lesser increases in aspartic acid and some of the other amino acids are 
apparent in plasma. 
From this study it is evident that the lymphocytes from chronic lymphatic leuke- 
References p. 447/448 
