FREE AMINO ACIDS OF BLOOD AND URINE 
N 
No 
Qo 
Sample preparation 
Blood plasma. Fasting blood samples (50 ml) are collected in a plastic centrifuge 
tube, directly from a silicone-coated (Arquad 2 C, Armour and Co.) large gauge 
needle, on dry powdered anticoagulant (sodium polyethanolsulfonate, “Liquoide” 
Roche, F. Hoffmann, La Roche and Co, Basel, Switzerland). An alternative pro- 
cedure is also described below. The plasma is immediately separated by centrifu- 
gation at 5000 x g for 10 min. Two to-ml aliquots of plasma, numbered 1 and 2 
according to order of sampling, are deproteinized at once by 50 ml of 1% picric 
acid solution, according to the method of HAMILTON AND VAN SLYKE!®, and cen- 
trifuged. From the supernatant of each sample, two 25-ml aliquots are separately 
processed by the desalting method described by STEIN AND Moore”, if the automatic 
procedure is to be used. In the case of the manual procedure a 50-ml aliquot is re- 
quired. After desalting, concentration and adjustment to pH 2.0, the two samples, 
equivalent each to 4.16 ml of the original plasma, will be suitable one for acidic 
and neutral amino acid separation and the other for basic amino acid separation in 
the double-stage automatic procedure. Aliquots prepared from sample No.1 will 
be preferentially used for analysis since, being taken from the upper layer of plasma, 
they are probably free of light blood cells like platelets if any of these had failed 
to sediment. Recently, BRIGHAM, STEIN AND Moore have described a new procedure 
for blood-samples preparation which allows for the separate determination of cysteine 
and cystine concentrations in blood plasma", but this procedure has still not been 
applied in our investigations. 
Urine. As a rule urine analyses are carried out on aliquots of quantitative 24-h 
collections, irrespective of diet. In some instances 24-h collections have been frac- 
tionated in sub-collections of variable length of time when desirable for location of 
a peak excretion. During the 24-h collection, urines are collected in a one-gallon 
bottle containing 5 ml of toluene and 5 ml of chloroform as preservative. When 
collection is completed, total volume is recorded and an aliquot sufficient for repeated 
analyses is stored in a plastic vial and kept in the deep-freezer at —35° pending 
analysis. In some pathological specimen, when proteins were present, urine samples 
have been deproteinized by the method used for plasma samples!*. I or 2 ml of urine 
brought to pH 2.0 by HCl addition are used for analysis depending upon the 24-h 
urine output, the latter one being smaller or greater than 11. 
Blood ceils and plasma. Separation of the four constituents of blood in a pure form 
is by no means an easy task, especially when dealing with small volumes of blood. 
The following procedure, slightly modified of that of TRuHAuT!S, has been used, 
but, as will clearly be seen from discussion of analytical results, suffers from certain 
drawbacks, which are to be avoided if possible: 
I) 100-ml blood collection in unwettable centrifuge tube, using 5 ml of a 2% 
disodium EDTA solution in saline as an anticoagulant. 
2) Separation of plasma by undelayed centrifugation at 5000 x g for 45 min 
in the refrigerated centrifuge at 4°. Deproteinization of two ro-ml aliquots of plasma 
is performed as described under Blood plasma. A correction factor, owing to dilution 
of the blood by EDTA solution has to be calculated. 
References p. 261/262 
