352 G. ROUSER, B. JELINEK, A. J. SAMUELS, K. KINUGASA 
The marked variability in the results reported from different laboratories appears 
to be due in part to the relatively small number of samples examined so that a 
broad picture of the factors that influence the free amino acid levels in cells and body 
fluids was not obtained. The large number of samples to be examined in our own 
studies precluded the use of any but simple, rapid methods. It is for this reason that 
we chose semi-quantitative examination by paper chromatography. When samples 
are carefully prepared under controlled conditions and frequent comparisons are 
made between samples and known mixtures, reproducible results can be obtained 
and patterns or levels of free amino acids may be determined without giving the 
levels a numerical designation. Such studies should include photographs of the paper 
chromatograms so that the reader may make his own comparisons and draw his 
own conclusions. Paper chromatography was selected in preference to column- 
chromatographic procedures because the column procedures are very time consuming, 
there is usually some fraction overlap, glutamine is not recovered quantitatively 
from columns presently in use, and glutamic acid values may be in error. As we were 
particularly interested in possible changes in glutamic acid and glutamine, the latter 
objections are particularly important ones. 
The present report describes the general methods of study used in our investigations 
in which 2385 two-dimensional paper chromatograms were prepared from 34 blood 
samples and 18 urine samples from normal individuals; 12 blood and 6 urine samples 
from patients with acute leukemia; 174 blood samples, 23 urine samples, and 15 
bone marrow specimens from patients with chronic lymphatic leukemia; 199 blood 
samples and 20 urine specimens from patients with chronic granulocytic leukemia; 
59 blood samples and 70 tissue samples from rabbits; and 7 blood samples from 
leukemic and normal dogs. 
METHODS 
Separation of blood cells 
Blood from fasted individuals (12-18 h since the last meal) was drawn into heparin 
(0.5 mg/ro ml of blood) and centrifuged immediately. The following centrifugation 
technique was used for high count bloods. Blood, usually 10-12 ml, was placed in 
a 12-ml graduated centrifuge tube and the speed of centrifugation gradually in- 
creased to 500 x g over a period of 1-3 min, and centrifugation was continued at 
this speed for 7 min. After the initial spin, platelet-rich plasma overlying the white 
cells and red cells was withdrawn and transferred to another tube for recentrifugation. 
The white cell layer was next withdrawn with a small amount of blood plasma and 
placed in another 12-ml centrifuge tube for recentrifugation. Erythrocytes were with- 
drawn by inserting a needle to the bottom of the layer and aspirating the lower two- 
thirds of the cell mass (to minimize contamination with white cells) and placed in 
another 12-ml tube for recentrifugation. All transfers were made with a syringe 
equipped with a stainless steel needle 4 in. long that was rinsed with saline after each 
use. 
The relatively large white cell layer obtained from high count leukemias was 
recentrifuged at 1300 x g for 7-I0 min in a 12-ml centrifuge tube. The packing at 
this force gave reproducible volume measurements for white blood cells. Where the 
mass of leukocytes was small, a series of 10-ml tapered tubes was used for recentrifu- 
References p. 447/448 
