FREE AMINO ACIDS IN BLOOD. I 353 
gation of white cells at 1300 x g. The tapered portions of the tubes had volumes 
of 0.1, 0.4, or I ml and were calibrated in small units. These tubes facilitate accu- 
rate volume determinations and withdrawal of contaminating platelets and/or red 
cells. 
Erythrocyte samples were recentrifuged at 1300 x g for 10 min, and plasma and 
any residual leukocytes were aspirated before the cells were extracted. The red cell 
samples were not washed since control studies indicated losses of amino acids into 
saline or buffer washes. Generally, plasma and cell samples were extracted with 
alcohol within 30-40 min from the time blood was withdrawn. 
The isolation of white blood cells from normal individuals was accomplished with 
a unit of blood centrifuged in 500-ml bottles. The blood was spun at about 1300 x g 
to pack leukocytes, the cells were pipetted off, and recentrifuged in 12-ml tubes. 
After the second centrifugation white cells could be separated from most of the con- 
taminating erythrocytes. A third centrifugation (1300 x g) gave white cells largely 
free of erythrocytes. Cells prepared in this way were over 95°, neutrophilic polymor- 
phonuclear leukocytes. 
Extraction and preparation of samples for chromatography 
Samples were treated with 3 volumes of 95°% ethanol and the precipitate that devel- 
oped after vigorous shaking was filtered off on a sintered glass filter. The residue was 
extracted 3 times with small portions of 80°, ethanol. The combined ethanol solu- 
tions obtained from white cells and blood platelets were applied directly to filter 
paper for chromatography. 
The ethanolic extracts from blood plasma, erythrocytes, and urine were evaporated 
to dryness in 20-50 ml beakers under infrared heat lamps in a current of air from 
a fan. The beakers were placed on bright aluminum foil to prevent overheating of 
the samples. Next, the samples were dialyzed to remove lipid. The dry solids were 
treated with a measured volume of distilled water, transferred to well-washed Nojax 
cellulose casing and dialyzed against a measured volume of water for 4 hours at room 
temperature with constant shaking. The dialysis tubing was first washed by boiling 
twice for 30 min in distilled water to remove impurities that would otherwise interfere 
with the chromatographic examinations. The true aliquot size of the dialysate was 
determined from the ratio of water volumes inside and outside the bag. The dialysate 
was then desalted in a Reco electrolytic desalter with a 10 ml well at 20 mV for 5 to 
10 min. Usually samples equivalent to 3 ml of original plasma, erythrocytes, or urine 
were treated for 5 min. The desalted extract was then evaporated to dryness as de- 
scribed for ethanol extracts. 
Paper chromatography 
Untreated sheets of Whatman No. 1 filter paper for chromatography (18!/, x 221/, in.) 
were used. The dry solids from the desalting step were taken up in a measured volume 
of water and an equivalent of from 0.1~-1.0 ml of original sample (plasma, urine, 
erythrocytes) applied to one corner of the paper. A spot 3 cm in diameter is con- 
venient. When hydrogen peroxide was used to oxidize sulfur-containing amino acids, 
a solution of 30%, hydrogen peroxide (Superoxol) was spotted over the point of 
application of the sample. Rapid evaporation of the applied solutions was achieved 
by a current of air from a hair dryer. 
References p. 447/448 
