FREE AMINO ACIDS IN ANIMAL TISSUE 293 
tissues of various species were compared with the hydrolysates of the extracts. It is, 
of course, likely that small amounts of peptidic material would escape detection by 
the procedures employed. 
An extensive series of experiments then showed that the amino acid patterns 
observed in the freshly fixed tissues are in no way related to those found after autolytic 
changes have been allowed to take place??. Brain, muscle, liver and kidney were 
removed from normal Sprague-Dawley rats or those which have been hypophy- 
sectomized or adrenalectomized, as well as from tumor-bearing hosts. The Walker 
carcinoma was also studied under the conditions to be described. Results will be 
described in this report only for tissues of the normal rats. In order to insure that 
20 



p10, 
5 _ 
, i 
me 29 ie 17 re 120 
Figs. 29, 30. Influence of acid hydrolysis on distribution of constituents in protein-free extract 
of left ventricle of dog heart (75 mg). Fig. 29: unhydrolyzed extract. Fig. 30: same aliquot of 
extract after hydrolysis with 6 N HCl in a sealed tube for 24 h. Taurine, 5; alanine, 8; histidine, 
11; f-alanine, 12; glutamine, 13; glycine, 14; glutamic acid, 17; cystine (cysteic acid), 20; gluta- 
thione (oxidized with H,O,), 21. 
21 



17 
all changes observed would be attributable to enzymes found in the tissues, care was 
taken to exclude microorganisms. In an effort to avoid mixing substrate and en- 
zymes not ordinarily in contact with each other, pieces of intact tissue were used 
rather than homogenates, minces, or slices. The tissues were removed observing 
sterile precautions and samples were placed in individual sterile weighing bottles 
of known weight. After weighing, samples were placed in an incubator at 38° for 
varying periods of time. Upon removal from the incubator, cultures were made in 
thioglycolate broth from a small particle of each sample and the remainder of the 
sample was then worked up for chromatography in the usual manner. Results were 
used only for samples showing no microbial contamination. Virtually identical 
results were obtained for tissues of normal rats as were found for their hypophy- 
sectomized or adrenalectomized litter mates. The results at various time intervals 
after removal of the samples from the animals showed that the pattern of proteolytic 
breakdown is characteristic for each tissue. 
The results for various intervals of autolysis of brain up to 96 h after removal of 
the samples from the animals showed that extensive overall proteolysis had not 
taken place (Figs. 31-38). The amino acids were not liberated in the same relative 
amounts as during acid hydrolysis of brain protein. The changes in free amino acid 
content probably were the results of some processes by which the individual amino 
acids were formed or liberated and used during the period of observation. Since 
GABA is found chiefly in the easily extractable form in brain, the relatively large 
References p. 348/349 
