THE INFLUENCE OF CERT.AIM ENDOCRINE SECRETIONS 
m AMINO ACID OXIDASE* 
by 
Ralph N. Cagan, Capt., M.C., John L. Cray, Biochemist and 
K. Jensen, Ph.D., Chief Biochemist 
from 
Medical Department Field Research Laboratory 
Fort Knox, Kentucky 
16 Lay 1949 
under Study of the Physiological Effects of Cold* Approved 
24 Sept. 1942. M.D.F.R.L, Project No. 6-64-12-02-(9). Project No, 6-64-12-02 
Sub-project MDFHL 02-(9) 
MEDEA 
16 1949 
ABSTRACT 
THE INFLUEN CL OF CERTAIN ENDOCRINE SECRETIONS 
ON AMINO ACID OXIDASE 
OBJECT 
In the course of studies designed to investigate the effects of 
stress (hypothermia, shock and irradiation) on the amino acid oxidase 
system of the liver and kidney, it became necessary to know the influence 
of the blood amino acid level and of certain endocrine secretions on the 
activity of this enzyme system. Alterations in the oxygen uptake, as 
measured by the Warburg manometric technique, were used as indices of the 
changes in the enzyme activity. 
RESULTS AMD CONCLUSIONS 
Intraperitoneal administration of an amino acid mixture (casein 
hydrolysate) to normal rats produced an enhancement of the amino acid 
oxidase activity in the liver and kidney. Accelerated amino acid oxida- 
tion was simultaneously associated with increased blood urea and frequently 
increased glucose levels. In adrenalectomiaed and hypophysectomized ani- 
mals, similarly treated, the liver amino acid oxidase activity was not 
augmented. Administration of an adrenal cortical extract to normal and 
adrenalectomized animals accelerated the activity of the enzyme in the liver 
and kidney, An accelerating effect of the secretion of the adrenal cortex 
was also observed in vitro. The increase in enzymic activity of the liver 
observed in normal animals after amino acid administration is apparently 
mediated through the pituitary-adrenal cortex system. The nature of the 
mechanism of the? stimulation remains a matter for investigation. 
The pituitary may also inhibit liver amino acid oxidase activity by 
way of its growth hormone, for the liver of hypophysectomized animals showed 
increased amino acid oxidase activity. 
Thyroideeternized animals showed a decreased amino acid oxidase activity 
of the liver but an increased oxidase activity of the kidney. Administration 
of amino acids led to a stimulation of the liver oxidase. 
Epinephrine was found to inhibit amino acid oxidase activity of the 
liver. The effect of insulin was doubtful. 
The results obtained also indicate that while the activity of the liver 
amino acid oxidase is mainly under endocrine control, the kidney araino acid 
oxidase activity, at least partly, can be influenced directly by the amino 
acid level in the blood* RECCimiDATIONS 
Similar experiments should be carried out -with castrated animals in 
order to determine the regulatory effect of gonadal secretions on the amino 
acid oxidase activity. Additional studies should be carried out with hypo- 
physes tomized animals. 
It also is suggested that the possibility of an amino acid tolerance 
test be investigated in cases of either liver or certain endocrine 
(pituitary-adrenal cortex) insufficiencies. It is postulated that amino acid 
metabolism in patients with liver disease or certain endocrine (pituitary- 
adrenal cortex) deficiencies might be sufficiently impaired to decrease 
significantly the rate of disappearance of the amino acids from the blood. 
If such proved to be the case, the rate of removal of intravenously admin- 
istered amino acids may serve as an index of the degree of the liver or pitui- 
tary-adrenal cortex disfunction. 
Submitted by: 
H, h. Cagan, Capt., M.C. 
J, L. Gray, Biochemist 
H. Jensen, Ph.D,, Chief Biochemist 
/CtLij f/Ad q 
Approved: 
RAY ijU 
Director of Research 
Approved 
FREDERICK' J ./KNOBLAUCH 
Lt. Col«f *“ • c • 
Commanding THE INFLUENCE OF CERTAIN ENDOCRINE SECRETIONS 
ON MONO ACID OXIDASE 
I. INTRODUCTION 
The problem of intermediate protein metabolism has been studied for 
many years. Research during these years has answered many of the questions 
but many still remain unanswered. 
It is known that amino acids can either be converted into body protein, 
"anabolism”, or can be deaminated to form energy producing intermediates, 
"catabolism". The first step in the catabolic process is the deamination 
of amino acids. This reaction is catalyzed by the enzyme, amino acid 
oxidase, MAAO". There are two distinct amino acid oxidase systems (l): 
(a) 1-amino acid oxidase which specifically deaminates 1-amino acids and 
(b) d-amino acid oxidase which specifically deaminates d-amino acids. 
The activity of amino acid oxidase may be considered a measure of 
amino acid catabolism. The reaction is as follows: 
R - CK - COOH -f i Or, ► R - C - COOH -f NH~ 
I » 
nh2 0 
It may be observed that ammonia results from this reaction. In the liver, 
the ammonia participates in the formation of urea and in the process of 
transamination. The deamination of amino acids in the liver is accompanied 
by an increase in the urea of the blood. Changes in the level of blood 
urea therefore may be used as a partial index of the amino acid oxidase 
activity. 
It was the object of this study to investigate the interrelation of the 
influence of amino acid concentration in the blood and the secretion of the 
anterior pituitary, adrenal and thyroid glands on the rate of activity of 
the liver and kidney amino acid oxidase systems. Alterations in the oxygen 
uptake, as measured by the Warburg manometric technique, were used as 
criteria for changes in the enzymic activity. 
H. EXPERIMENTAL 
White male rats of the Sprague-Dawley strain weighing between 250-300 
grams were employed. They were starved for 18 hours before the experiment, 
but were permitted water. Four groups of animals were employed: normal, 
adrenalectomized, thyroidectomized and hypophysectomized. Within each group 
a series of experiments was carried out in each of which two animals were 
injected intraperitoneally with a 10 per cent solution of an enzymic casein 
hydrolysate* while one animal was simply pierced with a needle. For purposes 
of convenience, the latter procedure will be called "dry needle". 
* Amigen, prepared by Mead Johnson & Company, Evansville, Indiana, We 
wish to express our appreciation to Dr, Warren Cox of that company 
for generously supplying us with this preparation. 
1 Hie liver extracts were prepared by cutting out approximately three 
grams (weighed to the nearest 0*1 of a milligram) of tissue from the 
animals (killed by decapitation) and by homogenizing the tissue with 6 ml. 
of phosphate buffer (pH 7*35) for five minutes/ The buffer employed was 
a modified Krebs* buffer containing NaCl (0.95 per cent), MgSO, (3*32 per 
cent), KC1 (l,15 per cent) and made to pH of 7.35 with HCl and Na2HP0/+. 
After homogenization, the minced-tissue mixture was centrifuged at high 
speed for ten minutes. The supernatant was decanted and 2 ml. portions were 
used for the enzyme activity assay. Kidney was processed in a similar manner 
with approximately 1,2 grams of tissue being homogenized with 10 ml, of the 
same buffer for ten minutes. Samples of each tissue were taken to deter- 
mine the ration of wet to dry weights so that correction could be made for 
variation in water content of the tissue. 
It was necessary to attain a constancy of the tissue processing condi- 
tions so that the enzyme activity would vary only with the animal and its 
experimental environment. Accordingly, a specific number of minutes was 
allotted from the time the animals were killed until the tissue was homog- 
enized and from homogenization until the first readings were taken. 
The effect of amino acid injection on AAO activity was determined after 
periods of | hour and lj- hours from the time of injection. Animals were 
sacrificed at these time intervals and their tissue processed as related. 
In the Warburg manometric technique, employed for the determination of 
oxygen uptake, the flasks were filled as follows: 
Main Chamber - 2 ml. of buffered supernatant homogenate. 
Side Arm - flask - 0.2 ml. of a 0.1 molar 
dl-alanlne solution in buffer. 
- Control flask - 0,2 ml. of buffer. 
Central Well - 0,2 ml, of a 10 per cent sodium hydroxide 
solution. 
After replacing the air in the flasks with oxygen, they were placed in the 
water bath and equilibrated for 10 minutes at 36.8°C, 
After tipping the substrate into the main chamber, readings were taken 
every fifteen minutes for an hour with the flasks shaking 30 times per 
minute. The oxygen uptake is expressed in raicroliters of O2 per gram of wet 
tissue-homogenate extracted. 
The in vitro and in vivo experiments have been carried out in a sirailar 
manner in order to determine the effects of insulin, adrenal cortical 
extract and epinephrine on the AAO system. 
for the jja vivo experiments: (a) 0,5 I,U, of crystalline Insulin (Squibb) 
was injected intramuscularly, (b) 1 ml. of aqueous adrenal cortical extract 
(Upjohn) was injected hourly* intramuscularly, into the respective animal during 
a four hour period prior to sacrificing, or (c) 0,3 ml. of a 1:1000 adrenalin 
hydrochloride solution (Parke-Davis) was injected in the same manner as b, 
depending on the experiment. For the corresponding in vitro experiments, 0,05 
I.U. of insulin, 0,1 ml. of adrenal cortical extract~TUpjohn) or 0.1 ml. of 1:25,000 commercial adrenalin solution was used in each of the control and 
the experimental flasks which were prepared otherwise as described previously. 
Control determinations were run for each group of the in vivo and in vitro 
experiments* 
Blood glucose (2), urea (3), amino acids (4) and hematocrit determin- 
ations were carried out simultaneously on blood obtained at the time the 
animals were decapitated. 
Animals were adrsnalectomized from a single horizontal incision, posteri- 
orly in the lumbar region, four days, these animals were kept on a 
normal diet but given 1 per cent saline as drinking water. For the subsequent 
three days, they were taken off saline and given plain water and for the 
last eighteen hours, they were starved. These animals averaged a 25 gram 
weight loss from operation to day of experiment. 
Hypophysectomized animals were operated via a para-tracheal approach 
and were kept 14 to 21 days on a milk, chopped meat and bread diet before 
the experiment. This group had its own control group fed on a similar diet. 
The hypophysectomized animals averaged 50 grams less in weight than their 
litter mate controls at the time of sacrifice. 
Animals were thyroidectomized and maintained for at least 21 days before 
being subjected to experimental procedures. Only those animals that evinced 
a minimum of 25 per cent decrease in B.M.R. were used. The average lowering 
of B.M.R, among the twenty-four animals used was 35*2 per cent (see Table 1). 
TABLE 1 
BASAL METABOLIC RATS IN THYROIDECTOMIZED AND NORMAL ANIMALS 
Calories 
Per Square Meter 
Per Hour 
Number of 
Animals 
% Reduction 
Normals 
38.1 f 3.0 
9 
Thyroidectoinized 
24.7 i 1.83 
24 
35.52 
Another group of animals was operated (neck dissection) in a sham 
fashion to determine whether any effect on the amino acid oxidase resulted 
from the operation alone. No changes were found when the results were 
compared with those of unoperated control animals. 
III. RESULTS AND DISCUSSION 
A, Liver Amino Acid Oxidase 
From Table 2, it is obvious that administration of amino acids to 
normal animals caused a pronounced increase in the liver amino acid oxidase 
activity. However, neither adrenalectomized nor hypophysectomized animals 
similarly treated, showed this increase. The effect of the increased blood amino acid level on the liver amino acid oxidase is probably mediated 
through the pituitary-adrenal system. The nature of the "priming" mechanism 
for the pituitary-adrenal cortex stimulation after amino acid administra- 
tion still must be investigated. The question whether the elevated blood 
amino acid level causes a direct or indirect stimulation of the anterior 
pituitary cannot as yet be answered. Paschkis and Schwoner (5) postulated 
that the pituitary produces a "protein metabolism hormone", which is re- 
leased on stimulation by a protein meal and may be found in the urine. 
Thus, they conclude that protein itself furnishes its own trigger mechanism 
for its metabolism. 
The effect of the adrenal-cortical secretion on the oxidase activity 
of the liver was also observed by in vitro and jlji vivo experiments. Table 3 
illustrates that normal as well as adrenalectomized animals, given adrenal 
cortical extracts, showed an increased oxidase activity of the liver while 
untreated adrenalectomized animals have a decreased AAO activity (Table 4)• 
In vitro experiments also showed a similar accelerating effect of cortical 
extracts upon amino acid oxidase activity of the liver (Table 3). 
It may be observed in hypophysectomized animals that the amino acid 
oxidase activity of the liver was increased 100 per cent over that of normal 
animals (see Table 4)• This increase in activity is probably due to the 
absence of the pituitary growth factor and is in agreement with the concept 
that the growth hormone may inhibit amino acid catabolism. It has been shown 
by Szego and ’white (6) that growth hormone produces increased fatty acid 
metabolism and fat deposition in the liver when administered to normal starved 
animals. These investigators suggested that the growth hormone may either 
inhibit amino acid catabolism or accelerate fat metabolism. Our observations 
support the former postulate, for in our experiments the hypophysectomized 
animals showed an increased amino acid oxidase activity in the liver, 
Hussell and Capiello (7) recently reported that when a partially purified 
preparation of anterior pituitary growth hormone was given to nephrectomized 
rats, 1 to 2 hours before the periods of observations were begun, the rate 
of urea formation during the first hour after the administration of a casein 
hydrolysate was reduced by approximately 40 per cent. 
In thyroideeternized animals, administration of amino acids resulted in 
an increase in amino acid oxidase activity of the liver which, however, was 
not as pronounced as in normal animals (see Table 2). According to Deane 
and Creep (ft), thyroidectoroy leads to an atrophy of the adrenal cortex. 
This may explain why the increase in liver amino acid oxidase activity in 
thyroidectomized animals is not os great as in normal animals. Thyroid- 
ectomized control animals showed a decreased AAO activity of their liver 
(Table 4). This finding is in agreement with that of Klein (9) who found that 
thyroidectomy decreases liver AAO activity. 
B. Kidney Anlno Acid Oxidase 
Strictly speaking, comparative results, as found for the liver amino 
acid oxidase, were not obtained for the kidney amino acid oxidase after amino 
acid administration (see Table 2). Values were obtained in Hie normal animal’s 
kidney showing an ft9 per cent increase in oxidase activity after hour but 
only a negligible increase after ij hours. A similar type of disagreement was also observed in the kidney amino acid oxidase activity of adrenal- 
ectomized and hypophysectomized animals. There is no increase after hour 
in the adrenalectomized animals but a 59 per cent increase in the amino 
acid oxidase activity after hours. Again, peculiarly, an increase in 
activity of 29 per cent occurs in the kidney of hypophysectomized animals 
after ij hours. However, in the thyroidectomized animals, the kidney amino 
acid oxidase activity was distinctly decreased after amino acid,administra- 
tion, This inhibitory effect has still to be elucidated. 
Apparently, there is a distinction between the factors influencing amino 
acid oxidase activity of the liver and the kidney, Lang (ll) and Kochakian 
(12) found that liver and kidney amino acid oxidase enzymes do not respond 
similarly, Lotspeich and Pitts (13) reported that the excretion of ammonia 
in the urine of the dog is proportional to the plasma amino acid level. 
They concluded that the renal amino acid oxidase is concerned with the 
formation of ammonia by the kidney which process plays an important role 
in the regulation of acid-base balance. Other investigators (14) have also 
found that the administration of certain amino acids produces an increased 
excretion of ammonia in the urine of the dog. If the ammonia is considered 
as a measure of the activity of AAO, since it is a product of the reaction 
of the enzyme, one may theorize that the amino acid oxidase activity in the 
kidney not only is dependent upon the various aforementioned endocrine 
factors, but may also vary in accordance with the amino acid level of the 
blood. Thus, the initial increase (Table 2) | hour after injection and 
subsequent decrease after hours in AAO activity of the normal animal's 
kidney may be attributed to the corresponding rise and fall in amino acid 
level of the blood. 
This view may similarly apply for the adrenalectomized and hypophy- 
sectomized group where the findings are reversed, i.e,, the greater increase 
in the AAO activity is attained at the hour period. In these instances, 
since the liver oxidase activity, in the absence of the activating mechanism 
of the pituitary-adrenal cortex system, cannot partake normally in lowering 
the amino acid level any longer, the effect of the continued blood amino 
acid elevation manifests itself in the kidney but not until a period of time 
has elapsed. 
Russell and Wilhelmi found (10) that the kidneys of adrenalectomized 
rats showed a decreased AAO activity, and that administration of adrenal 
cortical extract to these animals increased AAO activity. Our control adrenal- 
ectomized animals which were injected with a "dry needle0 did not show this 
decrease in 30 minutes after "injection0 but did manifest a decreased AAO 
activity below that of normal animals in hours after injection with "dry 
needle11, (see Table A.) 
A possible cause for this discrepancy will be discussed later when the 
effect of epinephrine on the AAO system is discussed. 
C, Blood-Amino Acid. Glucose, Urea Hematocrit (see Table 5). 
AAO activity may be correlated with the level of amino acid nitrogen, 
urea nitrogen and glucose in the blood. In normal animals (see Table 5), the 
amino acid nitrogen rose from 12.1 mg. to IB,5 mg, per cent in hour after 
5 injection of the amino acid mixture and declined to 15*4 mg. per cent in 
hours indicating rapid deamination. The changes in the blood amino acid 
nitrogen level can be linked with the respective changes in urea nitrogen 
and glucose levels of the blood. While urea nitrogen was not greatly- 
changed, 4*7 mg. per cent after J hour, it was elevated by 12.5 mg. per 
cent in ij hours after amino acid injection. Similarly, in normal animals, 
there was no increase in blood glucose in J hour but a 10.8 rag. per cent 
Increase after l| hours. These results probably indicate that the increased 
deamination of amino acids led to an increased formation of ammonia and 
consequently of urea and that simultaneously increased gluconeogenesis had 
taken place. Acceleration of these metabolic processes in normal animals 
had started in J hour and were well established in ij hours after the 
injection of -the casein hydrolysate. 
In the adrenalectomized animals, the increase in these transformations 
seemed to be slowed down or inhibited. For example, there is no decrease 
in amino acid nitrogen blood level in ij hours (20,5 mg, per cent in \ hour 
and 22,3 mg. per cent in hours) after the injection of the amino acid 
mixture. These amino acid nitrogen levels were distinctly higher than 
those found in normal animals (18,5 and 15.4 rag, per cent respectively). 
Apparently, the adrenalectomized animals are unable to accelerate properly 
the metabolism of the injected amino acids. These findings are in agreement 
with the generally accepted assumption that certain factors secreted by the 
adrenal cortex enhance protein catabolism. 
Furthermore, the increases in blood urea hour, 7.9 mg, per cent; 
li hours, 8,3 mg. per cent) and glucose (J hour, minus 10,5 mg. per cent; 
hours, minus 20 rag, per cent) of the adrenalectomized animals after the 
injection of amino acids are, with the exception of urea at J hour, not as 
great as compared to the increases in the same blood constituents of normal 
animals (urea, 4.7 mg. per cent and 12,5 mg, per cent; glucose, minus 6.3 mg. 
per cent, plus 10,8 mg, per cent) subjected to the same treatment (see Table 
5). The observation that one gets a significant increase in urea formation 
at all in the adrenalectomized animals may be surprising at first thought, 
since the lack of adrenal cortical secretion would lead one to believe that 
little or no deamination takes place. However, it must be recalled that 
the ability of the kidney to dearainate was not found to be as impaired as 
that of the liver in the adrenalectomized animals (see' Table 2). Thus, 
ammonia formed in the kidneys of these operated animals may be utilized in 
the liver for the formation of urea. In addition, the impaired excretory 
capacity of the kidneys in these animals may account for retention of some 
urea and this may account for the apparently greater increase in blood urea 
after J hour. 
In hypophysectomized animals, the amino acid nitrogen level, hours 
after amino acid administration (16,0 mg, per cent) is at the same level 
found in normal animals similarly treated (see Table 5). The control animals 
in the hypophysectomized group had an amino acid nitrogen level comparable to 
the normal controls (l2,5 mg. per cent). The ability of the 
animals to dearainate injected amino acids at a rate comparable to that observed 
in the normal animal may be due to the absence of the pituitary growth factor 
as noted previously. Furthermore, the absence of this factor may explain the 
6 apparently accelerated protein catabolism as shown by the findings that 
blood urea nitrogen is increased 12*6 mg. per cent (46,8 to 59*4) and 
glucose 16,6 mg. per cent (45*5 to 62.1) during this period (see Table 5). 
The increases are about the same as those found in normal animals after 
injection, and are greater than those values found in adrenaleeternized 
animals. However, the absolute values for blood urea are increased and for 
glucose are decreased in the hypophysectomized animals. 
By the same criteria, thyroidectomized animals manifest the ability, 
although somewhat retarded, to metabolite amino acids. From Table 5> it may 
be observed that the lowering of the amino acid nitrogen level was not quite 
as prompt or as effective but eventually, after injection of amino acids, 
the amino acid nitrogen level decreased from 21.7 mg. per cent in \ hour to 
16.0 rag. per cent in hours. Similarly, the urea nitrogen levels, although 
increased hour, 28.1 to 31.7 mg. per cent; hours to 39.3 mg. per cent) 
do not quite attain the increase that the normal animals showed in | hour 
and ij hours. Finally, the blood glucose levels in these animals increased 
in hour (3.9 mg. per cent) but decreased greatly (12.6 per cent) in ij 
hours after injection. 
It can be noted from Table 5, that the hematocrit is increased about 
6 to 8 per cent in the injected norml animals. This finding may be explained 
by the relative dehydration caused by the transfer of water from the hypotonic 
environment of the vascular compartment into the hypertonic environment of the 
peritoneal cavity into which a 10 per cent solution of amino acids had been 
injected. It should also be noted that the adrenalectomized animals showed 
a greater increase in hematocrit (8 to 12 per cent) after injection, than did 
normals• 
D, General Observations 
From Table k> it can be seen that those control normal and thyroid- 
eeternized animals, which only ’were pierced with a "dry needle" J hour 
previous to being sacrificed, showed a decreased amino acid oxidase activity 
in the liver and kidney whem compared with animals sacrificed 1-J hours after 
injection. Since this decrease was not manifested in adrenalectomized animals 
similarly treated and since it is associated with increased blood glucose 
levels (Table 5), one may suspect that the secretion from the adrenal medulla 
may be responsible for the transient decrease in amino acid oxidase activity. 
Hie results of experiments with epinephrine both in vitro and in vivo support 
this possibility (see Table 3). *Vhen a non-commercial epinephrine solution 
containing only pure crystalline epinephrine was used, the inhibitory effect 
lasted only for 15 to 20 minutes. This difference in effect may be due to 
the presence of antioxidants in the commercial solutions. 
It is also conceivable that the above mentioned inhibition may be 
due to the action of insulin on the AAO, The increased blood sugar levels 
caused by an epinephrine reaction may result in an increased insulin secre- 
tion. Although certain investigators (15,16) have been able to show insulin 
inhibition of this enzyme, our technique was unable to demonstrate this effect 
either in vitro or i£ vivo. (See Table 3). iv. SUMMARY AND CONCLUSIONS 
1. Data have been presented relating blood amino acid level and 
certain endocrine secretions to the activity of amino acid oxidase in the 
liver and the kidney of rats. 
2. Administration of casein hydrolysate to normal animals produces 
an increase in the amino acid oxidase activity of the liver and kidney in 
these animals. 
3. Administration of casein hydrolysate to adrenalectomized or hypo- 
physectomized animals does not produce this increase in the amino acid 
oxidase activity of liver. 
U. Adrenal cortical extract accelerates the activity of this enzyme 
in the liver in vitro and in vivo and in the kidney in vitro. 
5. The piutitary-adrenal cortex complex mediates the stimulus for the 
acceleration of amino acid oxidase activity of the liver observed after 
amino acid administration. 
6. The nature of the mechanism of the pituitary-adrenal cortex stimu- 
lation after amino acid administration remains to be investigated. 
?• Livers of hypophyseeternized animals show increased amino acid 
oxidase activity which may be due to the absence of the growth factor of 
the pituitary. 
8, Thyroidectomized animals show a decreased amino acid oxidase 
activity of the liver but an increased activity of the kidney. Adminis- 
tration of amino acids stimulates the liver oxidase. 
9. Epinephrine Inhibits amino acid oxidase activity of the liver. 
The effect of insulin is doubtful. 
10. The amino acid oxidase activity of the liver and the kidney may 
respond similarly to certain endocrine stimuli. However, it appears that 
the blood amino acid level may influence the kidney oxidase activity 
directly but not that of the liver. 
V. RECOMMENDATIONS 
In order to complete the concept of the effect of endocrines on the 
enzyme, amino acid oxidase, it is suggested that further work be done with 
hypophyseeternized and castrated animals. These experiments should be 
carried out similarly to those described in this report. 
It is further recommended that an investigation on the tolerance of 
humans to amino acid administration be initiated. The experiments to be 
carried out on normal humans and on those with either adrenal or liver 
insufficiency. It has been related in this report that the metabolism of 
amino acids is inhibited in animals with adrenal deficiency. It is postu- 
lated that amino acid metabolism in patients with liver or certain endo-* 
crine. (hypophysis-adrenal cortex) disfunctions might be sufficiently impaired 
to decrease significantly the rate of disappearance of amino acids from 
the blood. If such proved to be the case, the rate of removal of intra- 
venously administered amino acids may serve as an index of either liver 
or hypophysis-adrenal cortex functions. 
VI. BIBLIOGRAPHY 
1* Blanchard, M,, et 1-Amino Acid oxidase of animal tissue, 
J. Biol. Chem. 421, 1944. 
2, Somogyi, M, D, Determination of blood sugar. J. Biol, Chem. 
106: 69, 1945. 
3. Van Slyke, D, D, and G. E, Cullen, Determination of urea by the 
urease method, J. Biol. Chem, £k: 417, 1916. 
4« Frame, E, G,, J, A, Russell and A, E. Wilhelmi, The colorimetric 
estimation of amino nitrogen in blood. J. Biol, Chem, 149: 255, 
1943. 
5, Paschkis, K, E, and A, Schwoner, Output of protein metabolism 
hormone of pituitary anterior lobe, .Endocrinology. S>6: 117, 
1940. 
6, Szego, C, M, and A, Yfoite. The influence of growth hormone on 
fasting metabolism. Endocrinology, 150, 1949. 
7, Russell, J,,A. and M, Cappiello, The effects of pituitary growth 
hormone on the metabolism of administered, amino acids in nephrec- 
tomlzed rets. Endocrinology, 333, 1949. 
8, Deane, H, W. and R. 0. Greep. Cytochemical study of adrenal cortex 
in hypo- and hyperthyroidism. Endocrinology. 243, 1947. 
9, Klein, J, R, Effect of thyroid feeding and thyroidectomy on the 
oxidation of amino acids by rat kidney and liver. J, Biol. Chem. 
128: 659, 1939. 
10. Russell, J. A, and A, E, Wilhelmi. Metabolism of kidney tissue in 
adrenalectomized rat. J, Biol, Chem, 137: 713, 1941. 
11. Lang, K, The effect of the level of protein intake on the activity 
of oxidation enzymes in the organs, Klin. Wchnschr, 24-25/55-56: 
868, 1947. 
12. Kochakian, C, D. and M. N. Bartlett. The effect of crystalline 
adrenal cortical steroids, di-thyroxine, and epinephrine on the 
alkaline and acid phosphatases and anginase of the liver and 
kidney of the normal adult rat, J. Biol, Chem. 176: 243, 1948, 
13. Lotspeich, W. D. and R. F, Pitts. The role of amino acids in the 
renal tubular secretion of ananonia, J. Biol. Chem, 168: 611, 
1947. 
9 14. Bliss, S, Increased excretion of tirinary araraonia in dog follow- 
ing intr tvenous injection of both natural and unnatural forms of 
certain amino acids. J, Biol, Chem, 2311 211, 1941. 
15. Bach, S, J. and E, G. Holmes, The effect of insulin an carbo- 
hydrate formation in the liver, Biochem, J. Jl: 89, 1937* 
16. Stadie, 'V, C,, F. D. Lukens and J, A. Zapp, Jr, Effect of 
insulin upon urea formation, carbohydrate synthesis, and respira- 
tion of liver of normal and diabetic animals. J. Biol, Chem. 
132: 393, 1940. i 
Tissue 
■■■» 
lonutes 
After 
Injection 
- . 
Normals 
Adrenale ctomized 
Hypophyse c t oraiz ed 
1 
Thyroide c toiaized 
LI VSR 
30 
14 = 50? 
0 = (£ 
9 = 33? 
90 
15 = 33? 
2-1$ 
-31 = -38? 
3 = 11? 
KIDNEY 
30 
119 = 89? 
~2U = -9^ 
— 
-43 — -14? 
90 
18 = 7% 
107 = 59? 
66 - 29^ 
-53 = -27? 
i 
1 
lilCROLITEHS AND % INCREASE III AMINO ACID OXIDASE ACTIVITY AFTER AMHIO ACID INJECTION 
Values Equal Microliters and % Increase in Microliters of Oxygen 
Uptake Per Gram of Tissue Homogenate Extract 
TADLS 2 TABLE 3 
EFFECT OF CERTAIN HORMONES ON AMINO ACID OXIDASE OF LIVER 
m VITRO AND IN VIVO 
JHCROLITERS OF OXYGEN UPTAKE 
Figures 
in Parent 
theses Equal Number 
of Animals 
Buffer 
Alanine 
Difference (AAO) 
Control (AAO) 
ADRENAL CORTICAL 
EXTRACT 
In Vitro 
300 
425 
125 4 is (6) 
59 1 S (14) 
In Vivo 
ena le c t oi -liz ed) 
301 
343 
42 ± 3 (2) 
21 f 3 (2) 
In Vivo 
"(Normals) 
219 
OTTO 
*• 1 / 
60 4 4 (4) 
27.5 4 1.5 (2) 
ADRENALIN 
In Vitro 
232 
257 
25 4 2.6 (5) 
59 ± 8 (U) 
In Vivo 
230 
253 
23 i 6 (4) 
41 4 4.5(20) 
INSULIN 
In Vitro 
242 
274 
32 4 9 (13) 
59 i & (14) 
In Vivo 
203 
246 
43 4 4 (3) 
57 ± 9 (4)  
Figures in Parentheses Equal Nuaber of Aniaals 
Tissue 
Time 
After 
Infection 
Ury Needle*’ 
Normals 
Adrenalectoaized 
Hypophysectomized 
Thyroidectoaized 
Buffer 
Alanine 
Oxidase 
Activity 
Buffer 
Alanine 
Oxidase 
Activity 
Buffer 
Alanine 
Oxidase 
Activity 
Buffer 
Alanine 
Oxidase 
Activity 
0 
211 
270 
59 i 8 (10) 
— 
■ — 
— 
— 
— 
— 
— 
— 
-- 
LIVER 
30 
. 208 
*235 
27 i 3.3 (8) 
197 
226 
29 i 6.1 (9) 
— 
— 
— 
205 
223 
18 * 2.0 (4) 
90 
235 
275 
40 i 4.4 (9) 
171 
196 
27 i 5.1 (5) 
204 
285 
81 * 9 (5) 
201 
228 
27 i 3.1 (4) 
KIDNEY 
30 
206 
341 
135 i 13.5 (6) 
197 
462 
265 1 17 (6) 
— 
164 
475 
311 I 18 (4) 
90 
220 
472 
252 i 21 (13) 
198 
380 
182 f 14 (5) 
*181 
412 
231 i 15 (5) 
201 
551 
350 f 21 (4) 
As Above 
After Injection of 5 cc. Aaigen Intraperitoneally 
LIVER 
30 
90 
226 
224 
267 
279 
41 i 4.5 (20) 
55 i 3.1 (21) 
225 
179 
249 
207 
24 * 4.3 (19) 
28 i 4.0 (7) 
200 
250 
50 i 5.7 (5) 
219 
239 
246 
269 
27 i 5.0 (8) 
30 i 4.3 (8) 
KIDNEY 
30 
90 
205 
207 
459 
477 
254 i 36 (19) 
270 i 31 (28) 
169 
187 
400 
476 
221 t 25 (14) 
289 i 20 (7) 
173 
470 
297 i 30 (9) 
195 
194 
463 
451 
268 f 22 (8) 
257 * 5 (8) 
TABLE I 
TOTAL RESPIRATION AND AMINO ACID OX1IASE ACTIVITY OF CONTROL 
Am SXFERBSKTAT, AI’B'ALS 
fcicroliters of Oxygen Uptake Per Gram of Rocogenized Tissue Extract Blood 
Constituent 
Minutes 
After 
Injection 
Figures in Parentheses Equal Number of Animals 
Normals 
Adrenalectomized 
Hypophysectomized 
Thyroidectomized 
Control 
Injected 
Control 
Injected 
Control 
Injected 
Control 
Injected 
HEMATOCRIT 
% 
- 30 
90 
52.5 4 0.6 U) 
52.A 4 0.4(4) 
55.A 4 1.0(8) 
56.5 4 1.5(6) 
55.5 4 1.3(3) 
55.5 f 0.8(3) 
60.0 4 1.4(5) 
63.0 f 2.0(5) 
— 
— 
A9.A 4 1.5(A) 
50.5 4 0.9(3) 
51.2 4 0.8(8) 
55.0 4 2.0(6) 
AMINO AGH?S 
Mg. % 
Amino Acid 
n2 
30 
90 
12.1 4 1.3(5) 
12.9 4 1.4(9) 
18.5 4 1.6(8) 
15.A 4 0.2(12) 
12.6 4 1.3 (7) 
1A.5 4 0.6 (3) 
20.5 4 1.3(13) 
22.3 4 1.8(5) 
12.5 4 0.5(A) 
16.0 4 1.2(7) 
11.9 4 .9(3) 
12.2 4 .A(A) 
21.7 4 3.1(6) 
16.0 4 1.6(8) 
— 
UBEA 
Mg. % 
Urea N2 
* 
sO Vv) 
o o 
18.9 i 0.8(A) 
17.7 4 0.3(5) 
23.6 4 1.8(9) 
30.2 4 .3(8) 
A0.8 4 2.0(3) 
AC,3 4 2.6(3) 
AS.7 4 3.1(A) 
AS.6 4 2.8(2) 
46.8 4 2.2(3) 
59.A 4 1.6(6) 
28.1 4 0.9(3) 
29.1 4 1.5(3) 
31.7 4 1.8(7) 
39.3 4 2.1(5) 
GLUCOSE 
kg. % 
o o o 
<*■* 
68.2 | 0.2(3) 
72.9 4 2.1(3) 
66.0 4 3.0(5) 
66.6 4 2.4(8) 
76.8 4 2.7(10) 
A6.5 4 2.3(3) 
A7.8 4 2.0(3) 
36.0 4 6,1(5) 
27.8 4 1.1(5) 
A5.5 4 1.5(2) 
62.1 4 3.6(A) 
58.5 i 3.0(3) 
73.7 4 1.0(A) 
59.2 4 2.5(A) 
77.6 4 0.8(8) 
46.6 4 3.4(8) 
TABLE 5 
CHANGES IN BLOOD CONSTITUENTS DURING EXHiRIMENTAL PROCEDURES