452 E. V. FLOCK AND J. L. BOLLMAN 
evisceration. These tissues were also removed from normal rats. Plasma, brain, 
skeletal muscle, and the ventricular muscle of the heart were treated with picric 
acid, and the total a-amino nitrogen of the free amino acids in this filtrate was deter- 
mined by the method of HAMILTON AND VAN SLyYKE!’. Glutamine was determined 
by the method of HAmMILTon?®. 
For separation of the free amino acids on paper chromatograms, protein-free 
extracts of tissue were made essentially by the method of Sotomon ef al.1*. The 
extracts were desalted by the method of CONSDEN, GORDON AND MARTIN”? and two- 
dimensional chromatograms were developed by the ascending technic with the frame 
Skeletal muscle after evisceration 
s——Plus adrenalectomy—, 

} e=<—52° | 
>See 
With insulin 
----- Without insulin 
80 TF Total free amino acids 


(5 

Glutamine 
Mg «-NH, WN per 100 g 


Hours 
Fig. 2. Total free amino acids and glutamine in skeletal muscle of eviscerated rats and of evis- 
cerated-adrenalectomized rats. The groups of rats are the same as in Fig. 1. 
of Datta, DENT AND Harris?! as previously described? in a 75°% solution of phenol 
containing sodium cyanide and in an ammoniacal atmosphere, and then in a 65% 
solution of lutidine containing diethylamine. The sheets were sprayed with 0.25% 
solution of ninhydrin in ethanol, and the color was allowed to develop at room 
temperature for 3 h. The chromatograms were photographed or used for quantita- 
tion of individual amino acids. The colored spots were outlined lightly with pencil 
before the color development was complete. At the end of the 3-h period, the spots 
were cut out and placed in test tubes containing 50°, propanol, and allowed to 
stand except for gentle mixing for 30 min. The purple color was measured spectro- 
photometrically at 570 mw. Two-dimensional chromatograms of extracts of plasma 
and tissues were made with chromatograms of a standard mixture of amino acids 
under identical conditions. 
RESULTS 
Effect of time after evisceration on levels of amino acids in the rat. The content of free 
amino acids increased slowly and progressively in the plasma and the skeletal and 
References p. 460 
