EXCRETION IN REPTILES

I. NON-PROTEIN NITROGEN CONSTITUENTS OF THE

URINE OF THE SEA-TURTLE CHELONE MYDAS L.

BY FOUAD KHALIL
(From the Department of Zoology, Faculty of Science, Found Ist University,
Cairo, Egypt)

(Received for publication, July 30, 1947)

Very little is known about excretion in reptiles. Most of the earlier

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workers (1) occupied themselves with qualitative or approximate quanti-
tative determination of some of the constituents of the urine. Among all
these workers there is a general agreement that terrestial reptiles, except
the Chelonia, excrete mainly uric acid. On the other hand excretion in
the Chelonia is in dispute. In the case of land tortoises, Magnus and
Miiller (2) working on Testudo nigra, Marchand (3) on T. tabuZata, Schiff
(4) on T. tubulatu, Clementi (5) on T. gruecu, claimed the presence of urea
in the urine. Contrary to these authors, Mills (6), investigating the urine
of T. tubulutu, was unable to find urea and showed the presence of uric
acid. Drilhon and Marcoux (7) found in the urine taken from the bladder
of T. muuritunicu a considerable amount of uric acid and purines in sus-
pension and urea in solution. According to these authors almost all of
the non-protein nitrogen of the clear urine was in the form of urea. In the
case of aquatic turtles, Burian (8) working on Thulussochelysproved the
presence of uric acid in the urine. Lewis (9) and Wiley and Lewis (lo),
working on Chelone mydus and Chrysemys pi& respectively, found that
urea constituted the greatest part of excretory nitrogen as compared to
uric acid and ammonia.
In view of this controversy the present work was found necessary, es-
pecially since an investigation of someof the metabolic processesof reptiles
had already been undertaken.
Muterial and Methods
Only four specimens of Chelone mydus were obtainable, two males, Ani-
mals A and B, two females, Animals C and D. They were all nearly of
the same size and each weighed about 25 kilos. When the experiments
were conducted (in March and April), the females were found to contain
a large amount of eggs. The animals were decapitated and the blood
collected in sterile bottles, containing neutral potassium oxalate (0.2 gm.
per 100 cc. of blood). The animals were then opened, their bladders re-
611
612 EXCRETION IN REPTILES. I

moved, and the urine measured and kept in sterile bottles. The amount
of urine collected was as follows: Animal A, 175 cc., Animal B, 110 cc.,
Animal C, 260 cc., and Animal D, 65 CC.
The pH of the urine, estimated immediately by the British Drug Houses
capillator, was as follows: Animals A, B, C, and D, pH 4.4, 4.6, 4.4, and
8.5, respectively. The urine in all cases contained mucus and that of
Animals A and C showed a slight deposit. The urine was cleared from
mucus and deposit by centrifuging. Thymol was then added to the urine
(1 mg. per cc. of urine) and the urine was kept in the ice chest. Under
these conditions the pH remained unchanged.
A protein substance was found to be present in all specimens of urine.

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It was removed, when necessary, by heating the urine to boiling, acidifying
with dilute acetic acid, and filtering.’
Urine Analysis-The total non-protein nitrogen was estimated by the
Kjeldahl-Gunning method (11). Urea was estimated by the urease method
of Van Slyke and Cullen (12) with acetate buffer, the urease extract being
prepared according to the method of Damodaran and Sivaramakrishnan
(13, 14) from watermelon seeds. Ammonia was estimated both by Folin’s
(15) and the micro-Kjeldahl method (16). Uric acid was estimated by the
direct calorimetric procedure of Benedict and Franke (17) as modified by
Christman and Ravwitch (18). Amino acids were estimated by Folin’s
calorimetric method (15). The high concentration of ammonia in urine
necessitated the addition of 3 gm. of permutit for each 10 cc. of 1: 20 urine,
and the gentle agitation was prolonged for 15 minutes. This process was
repeated twice. Urine after such treatment contained no traces of am-
monia. Creatinine was estimated by Shaffer’s calorimetric method (19) ;
creatine by Folin’s microchemical method (20). Griffith’s method (21)
was used for estimation of hippuric acid, and Larson’s (22) for estimation
of allantoin.
The deposits present in the urine of Animals A and C were examined
microscopically and were found to consist of yellow uric acid crystals.
These deposits were washed with warm water and then dissolved in a 2
per cent solution of piperazine and estimated calorimetrically as mentioned
above.
Blood Anabysis-Protein-free blood filtrate was obtained from unlaked
blood by Folin’s method (23). The total non-protein nitrogen in the
1 The presence of such a protein substance was detected not only in these turtles
but also in a number of other reptiles already examined by the author. The presence
of this protein substance seems to have been overlooked by all earlier workers on the
Chelonia except Mills (6) who demonstrated its presence in all of the animals he
examined. The origin and significance of this protein substance are being investi-
gated.
 F. KHALIL 613

blood filtrate was estimated by the Kj eldahl-Gunning procedure (11). Am-

monia was estimated according to Nash and Benedict (24). Urea was
determined in the blood filtrate by urease hydrolysis, with acetate buffer,
aeration, and nesslerization (15). Uric acid was estimated by Folin’s
direct method (25, 26). Amino acids were determined by Folin’s pro-
cedure (27) as modified by Danielson (28) and Russell (29).

RESULTS AND DISCUSSION

The results of the analysis of urine are shown in Table I. Ammonia

forms the major excretory nitrogen end-product. This is in harmony with
what is known in aquatic invertebrates and marine teleostian fishes (30-

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TABLE I
Non-Protein Nitrogen Constituents of Urine of Chelone mydas
-
Nitrogen in 100 cc. urine Nitrogen partition, per cent of total
N excreted

Constituents Aver- AWX-

~iyl Agnimnl A;im;l Animal age, Animal Agniyl A;im;l Animal age,
D, 0 pgma; A, 8 , , D, P Animals
3 , A, B, C
--~--~~~--
ms. m&T. ms. ms. ms.
Total N 288.7 399 262.5 210 316.7 100 100 100 100 100
Ammonia N 93.8 201.6 120.4 61.6 138.9 32.49 50.52 45.87 29.33 42.96
Urea N None None None 24.7 None None None None 11.75 None
Uric acid N 4.2 7.5 8.8 13.2 6.8 1.45 1.88 3.36 6.27 2.23
Allantoin N 12.6 47.9 64.4 15.6 41.6 4.37 12.0 24.53 7.44 13.63
Amino acid pi 15.7 29.3 25.8 38.6 23.6 5.42 7.34 9.83 18.38 7.53
Hippuric acid K 66.5 42.0 21 31.5 43.2 23.04 10.52 8.0 15.0 13.85
Creatinine N 3.3 6.4 4.9 3.5 4.8 1.14 1.60 1.87 1.67 1.54
Creatine N 27.8 17.4 10.8 8.8 18.6 9.63 4.36 4.12 4.18 6.04
Undetermined Ir 64.8 46.9 6.4 12.5 39.3 22.4 11.8 2.4 6.0 12.2

32). Ammonia is also present in the blood (Table II). The mode of
appearance of ammonia in the urine seemsto be quite different from what
is known to occur in mammals, in which ammonia is absent from the blood
(33) and forms a low proportion of the excretory nitrogen in the urine. In
this latter case, its formation and quantity are conditioned by the reaction
gf the urine. It is formed almost entirely in the kidneys (3436), mainly
from glutamine (37) and also from amino acids but not from urea (38,39).
As to the sea-turtle, although the study of the formation of ammonia is
not complete, the data so far available seem to throw some light on this
problem. From Table II it is seenthat urea is present in the blood of all
specimensexamined, while Table I shows that it is absent from the urine
of Animals A, B, and C and present only in the urine of Animal D (Table I).
614 EXCRETION IN REPTILES. I

The percentage of excreted ammonia nitrogen in Animal D is decidedly

lower than the average for the other three animals. On the other hand
the sum of the percentages of ammonia nitrogen and urea nitrogen of this
animal comes very near to the average percentage of ammonia nitrogen
for the other three animals. This, added to the fact that the blood of all
the specimens examined contains an appreciable amount of ammonia as
well as urea, suggests that the ammonia in the urine of the sea-turtle might
originate partly from blood ammonia and partly from blood urea. This
suggestion receives further support from the fact that the higher percentage
of excretory ammonia in Animals A, B, and C is not accompanied with

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TABLE II
Some Non-Protein Nitrogen Constituents of Blood of Chelone mydas
Nitrogen in 100 cc. blood
Constituents
Animal A Animal B Animal C Animal D

w. ms. w. mg.
Total non-protein N ................... 108.7 560 280 420
Ammonia N ........................... 11.2 3.8 6.9 3.8
Urea N ................................ 25.5 40.4 16.7 14.2
Uric acid N ............................ 6.0 1.6 2.1 1.6
Amino acid N ......................... 15.9 9.2 15.0 9.8

TABLE III
Uric Acid As Deposit in Urine

Total uric acid Uric acid N Uric acid N in 100 cc.

Animal urine
I I
WT. ntg. m?.
A 45.5 15.17 8.67
C 79.8 26.6 10.23

decreasedamounts of excretory amino acids. The part of ammonia which

originates from blood urea is probably formed in the kidneys.
Table I also shows that the main part of the end-product of purine
metabolism is in the form of allantoin and a small part in the form of uric
acid. But it looks as if the ability of turtles to oxidize uric acid to al-
lantoin is less than that of mammals, as the values of uricolytic index of
these animals are as follows: Animal A 75, Animal B 86.5, Animal C 88,
and Animal D 54.2. Moreover if the uric acid, which is found as a deposit
in the urine of Animals A and C (see Table III) and which should be in-
cluded with excretory uric acid, is taken into consideration, the values of
the uricolytic index of these two animals will be 49.4 and 77.2 respectively.
 F. KHALIL 615

In mammals the uricolytic index is much higher. The average for dif-
ferent rodents is 95, for different ungulates 90.2, and for different Carnivora
97 (40). It is remarkable t,hat Wiley and Lewis (lo), using the method
of Christman, have failed to find allantoin in the urine of Chrysemys p&z.
Table I shows also that hippuric acid is excreted in large amounts. This
may be due to the herbivorous diet of these animals. Hippuric acid is
already known to occur in large amounts in the urine of herbivorous mam-
mals: horses (41), cows, oxen, and sheep (42).
The comparatively high concentrations of hippuric acid and creatine
excreted by the animals investigated recalls what is known to occur in
mammals (43, 44). Details of the mode of formation of these substances

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in sea-turtles are at present being investigated.
SUMMARY

1. Ammonia forms the major nitrogen end-product in the urine of Che-

lone mydas.
2. Urea is absent from the urine of the majority of the animals examined.
3. Allantoin forms the main end-product of purine metabolism. The
uricolytic index is, however, less than that in mammals.
4. Hippuric acid is found in the urine in appreciable amounts.

The author wishes to express his thanks to Professor K. Mansour and

to Dr. J. J. Mansour-Bek for valuable advice and criticism.
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