FREE AMINO ACIDS IN BLOOD. I a5 
On 
RESULTS 
Limitations of the method 
The limitations of the method are related largely to the presence in some samples 
of interfering substances of unknown nature not removed in the sample preparation, 
and to the limits of detectibility set by the ninhydrin reagent. In plasma and eryth- 
rocytes distinct spots of phenylalanine, tyrosine, histidine, arginine, and lysine 
are not usually observed except at aliquot sizes of the order of 0.5—1 ml. Phenyl- 
alanine and tyrosine are present in relatively small amounts while the ninhydrin 
color for histidine is somewhat less than for other compounds. The arginine and 
lysine spots are frequently poorly formed due to interference of migration from 
other substances. 
The overlap of spots may prevent the recognition of some compounds. Thus, 
leucine and isoleucine migrate together and the glycine and asparagine spots may 
overlap and merge to varying degrees with the serine spot in samples, even though 
these compounds in a standard mixture are usually well separated (see Figs. 1-10). 
A good deal of variation has been observed with paper obtained at different times. 
With some batches of paper glycine, serine, and asparagine were separated com- 
pletely from each other. On occasion paper was obtained that had the interesting 
quality of allowing wide separations of the pairs glycine-serine and alanine—threonine 
by virtue of a great increase in the migration of threonine and serine in lutidine without 
alteration of the migration characteristics of other amino acids. 
The variability of the filter paper gave rise to other limitations. Cysteine and 
cystine must be oxidized before they can be visualized on chromatograms. When 
samples are treated with hydrogen peroxide, variable destruction of amino acids 
may take place. This destruction may be very great on some filter papers and is 
particularly noticeable with plasma samples. The destruction of amino acids by 
peroxide was not as extensive with erythrocyte, leukocyte, or urine samples. The 
phenomenon made it impossible to examine some plasma samples for the sulfur 
amino acids. 
The various steps in the procedure for preparation of the samples were checked 
with authentic compounds and mixtures of standards and no alterations of any of 
the known substances were observed after dialysis or desalting. 
Compounds seen on two-dimensional paper chromatograms from blood and urine samples 
Table I gives the composition of an artificial plasma amino acid mixture prepared 
from pure substances with amounts in general to correspond to average values from 
the literature. Some adjustments were made upwards or downwards from the litera- 
ture values. Figs. 1-10 show the paper-chromatographic results obtained with differ- 
ent aliquot sizes of the artificial plasma amino acid mixture. These chromatograms 
show the variations in spot size and color intensity with different amounts of known 
substances and are of value for comparison with sample runs. Evidently the literature 
values are similar to the levels frequently seen in our studies. 
The free amino acids seen in plasma are: leucine plus isoleucine, valine, proline, 
alanine, glutamine, threonine, glycine, serine, taurine, glutamic acid, and cystine— 
cysteine (as cysteic acid after peroxide oxidation). Aspartic acid is frequently ob- 
served and at times a-amino n-butyric acid is also observed. Very occasionally, 
References p. 447/448 
