The Pathways of Nitrogen Excretion
in Littorina Planaxis
Robert Moulton
May 30, 1964
Hopkins Marine Station
Pathways of Nitrogen Excretion in
Littorina planaxis
In the kidneys and livers of animals having ureotelic mechanisms
of excretion, according to Clementi (1915; 1918), the enzyme arginase,
important in urea formation through the Krebs ornithine cycle, is
present in high concentration, whereas those which have a uricotelic
mechanism have a much lower concentration of this enzyme and
possess other enzymes such as xanthine oxidase which is important
in the formation of uric acid from purines synthesized from amino
acids. These organisms may also possess the additional enzymes
dricase and urease to degrade the uric acid which is formed from
the purine bases to urea and ammonia (Needham and Baldwin 1934). This
study will show that Littorina planaxis falls into the category
of uricotelic organisms since much of this organism's waste nitrogen
is first converted to uric acid. Estimations of specific enzymes
such as xanthine oxidase can give some insight into the possible
excretory pathways involved. In addition, since many organisms
continue the degradation of uric acid into simpler compounds before
excretion, tests for uricase and urease are of importance so that
a clear understanding of the overall excretory process may be
obtained.
Experimental Procedure
The essential basis of all the methods which have been employed
for the detection of arginase, uricase, urease, and xanthine oxidase
consisted in following the conversion of the substrates arginine,
uric acid, urea, and hypoxanthine respectively. In each test,
2-
approximately 25 kidneys or digestive glands were minced in artificial
sea water (McLeod, Onofrey, and Norris 1954) adjusted to pH 7.0
and then distributed among 10 test tubes in 0.5 ml aliquots.
Substrate was added and the activity of any enzyme which may have
been present was stopped by the addition of dilute sulfuric acid
and sodium tungstate solution, the final solution having a pH of
7.0. In each test, a set of controls containing tissue extract or
substrate alone were run simultaneously and treated in the same
manner as described above.
An arginine monohydrochloride solution, .85 M, was prepared
and the pH adjusted to 7.0 by the addition of 10% NaoH solution.
The urea substrate was prepared by dissolving 6 grams in 100 ml of
artificial sea water to make a.1 M solution. Uric acid, 16.8 grams,
was dissolved in 50 ml of artificial sea water to make a.2 M solution.
This solution was prepared fresh before each series of tests.
Hypoxanthine, 68 mg, was added to 10 ml of .05 M NaoH to make a
.85 M hypoxanthine solution at pH 8.3.
Ammonia was detected by Nesslerization (Haden 1923). Urea was
detected similarily after treatment with urease (Sumner 1926).
Uric acid was assayed by the method of Benedict (1924). All
colorimetric measurements were made in a Klett-Summerson Photo-
electric colorimeter.
Arginase activity was estimated by following the production
of urea from arginine. Xanthine oxidase activity was estimated
by following the production of uric acid from hypoxanthine.
Uricase activity was estimated by following the decrease in uric
acid. Urease activity was estimated by following the production of
-3 -
ammonia from urea.
Results and Discussion
The results of the experiment are presented in Table 1. Arginase,
an enzyme present in most terrestrial organisms, could not be detected
in any of the tests performed on Littorina planaxis indicating that
the formation of urea arises through pathways other than the Krebs
ornithine cycle. To account for the relatively large amounts of
urea excreted, the breakdown of uric acid seems to be an excellent
possibility.
It is possible that synthesis of less oxidized purine compounds
such as hypoxanthine and xanthine from amino acid nitrogen could
provide xanthine oxides. Xanthine oxidase would deal not only with
the nitrogenous compounds ultimately derived from nucleic acid
catabolism, but also with nitrogen derived from protein catabolism
as well. An active xanthine oxidase was found to be present in the
kidney tissue of this snail. Therefore, the majority of the uric acid
produced within the snail could be accounted for through the metabolism
of purine bases either ingested and degraded directly or synthesized
from other nitrogenous compounds.
Table 2 shows that the degradation of uric acid by way of uricase
leads to the production of ammonia. This conversion indicates that
allantoinase and allantoinase are present as well as the assayed urease.
These snails appear to have specialized more than many invertebrate
groups in the production of uric acid as a nitrogenous end product
as can be seen from the results of these tests and those of Needham
and Baldwins' (1934). These tests also show that further degradation
of uric acid does in fact occur with the help of the enzymes uricase
27
4 -
and urease which are both present and which are responsihle for the
two important end products, urea and ammonia.
The uricolytic cycle has been well worked out by Florkin and
Ducha'teau (Florkin, 1960). The first stage in the process of
uricolysis consists in the oxidation of uric acid itself to the
more soluble substance, allantoin, under the influence of uricase.
Allantoin is further degraded through a series of steps. The urea
produced in this eycle is further degraded by the action of urease
which is present in significant amounts in the snail Littorina
planaxis to give ammonia.
The majority of the conversions takes place within the kidney-
heart complex. The digestive gland tissue failed to show significant
enzymatic activity. Furthermore, the existence of these enzymes
in the kidney could possibly be an evolutionary adaptation allowing
the snails to produce excretory products according to the availability
of water. This mechanism may also be an intermediate step toward
terrestrial life.
Summary
1. An account is given of the experiments designed to elucidate
the sources and modes of formation of the excreted urea, uric acid, and
ammonia in the marine gastropod Littorina planaxis.
2. It is suggested that arginase is not concerned with the
production of urea in the members of this species because no correlation
was found between the amount of urea and arginase activity. In the
production of urea and ammonia, which represent the principal
nitrogenous end products of the Littorines, the enzyme arginase is
not of significant importance.
3. Xanthine oxidase is shown to present in the kidney of this snail,
though hitherto, it has not been found in other marine invertebrates.
(Baldwin and Needham 1934). It is suggested that this enzyme maybe
responsible for formation of uric acid in Littorina planaxis and other
members of this group, whether the uric acid is derived ultimately
from nucleic acid breakdown or from protein deamination.
4. The uricotelic character of the metabolism of this gastropod
is supported by the presence of urease, uricase, and xanthine oxidase
in the kidney tissue and indicate a mechanism which can account for
most of the nitrogen excretory products.
Table 1
All values in mg of substrate converted
Final
Initial
Incubation
Enzyme
time
Arginase
Argine control
.009
.010
16 hr
trial
.009
.006
16 hr
trial 2
Arginine-kidney
008
16 hr
.014
trial !
.018
.010
16 hr
trial 2
Kidney control
.010
.010
16 hr
trial
.009
.013
16 hr
trial 2
Arginine control
.009
.008
16 hr
trial !
.008
.007
16 hr
trial
Arginine-Dig. Gland
.015
16 hr
trial !
.012
.014
16 hr
trial 2
.012
Dig. Gland control
.011
16 hr
.010
trial
.008
16 hr
.012
trial

Xanthine oxidase
Hypoxanthine control
0005
8 hr
.0003
trial
.000
0006
8 hr
trial
Hypoxanthine-kidney
.0005
.0013
8 hr
trial
.0013
.0003
8 hr
trial2
Kidney control
.0002
trial
0001
8 hr
.0001
8 hr
.0002
trial
Hypoxanthine control
.0005
16 hr
.0007
trial!
0008
trial 2
16 hr
.0007
ypoxanthine- Dig. Gland
trial !
0006
16 hr
.001
.0005
.001
16 hr
trial 2
Digestive Gland control
000
trial
16 hr
.0007
.0007
trial 2
16 hr
.0005
Difference
+.001
+.003
-.006
+.008
.000
+.004
+.001
+.001
+003
+.002
+.001
+.004
+.0002
+ 0003
+.001
+.001
+.0001
+.0001
+.0002
+0001
+0004
+.0005
.000
+.0002
mo frainme
me gea
Table 1 continued
Enzyme
Initial
Incubation
time
Uricase
Uric acid control
trial 1
8 hr
.20
trial 2
8 hr
.20
ric acid-kidney
trial 1
8 hr
.19
trials2
19
8 hr
Kidney control
trial
0004
8 hr
trial 2
o004
8 hr

Uric acid control
trial 1
8 hr
.19
trial 2
8 hr
.20
Uric acid-Dig. Gland
trial 1
8 hr
.19
trial 2
.19
8 hr
Dig. Gland control
trial
8 hr
.0003
trial 2
.0003
8 hr
Urease
Urea control
8 hr
trial
008
trial 2
.007
8 hr
Urea-Kidney
8 hr
trial
.010
trial
8 hr
.007
Kidney control
trial
.008
8 hr
trial 2
8 hr
.009
Urea control
.008
8 hr
trial !
trial 2
8 hr
.006
Urea- Dig. Gland
trial 1
.020
8 hr
.017
8 hr
trial 2
Dig. Gland control
trial!
8 hr
.01
trial 2
.009
8 hr
Final
tion
.20
.20
.12
.12
.0005
0004
.19
.20
185
185
0004
0004
.01
.010
.056
.056
.01
.01
.01
.01
.019
.019
.012
.012
Difference
000
000
+.08
+.08
+.0001
+.0001
000
000
+.005
+.005
+.0001
+.0001
+.008
+ 012
+.184
+.196
+.008
+.004
+.008
+.016
-.004
+.008
+.008
+.008
nq Urie actd
ma a
Table 2
All values in mg of substrate converted
Production of ammonia from uric acid by uricase
Incubation Final NH,
Initial NH3
Enzyme
time
Uric acid control
.001
8 hr
trial !
.002
8 hr
.002
.001
tri,12
Uric acid-kidney
8 hr
.022
.004
trial !
8 hr
.005
trial 2
.025
Kidney control
.001
.003
8 hr
trial
.003
trial 2
.001
8 hr
Uric acid control
8 hr
.001
trial !
.001
8 hr
.002
trial 2
.001
Uric acid-digestive gland
.010
.005
8 hr
trial
.008
8 hr
.013
trial 2
Digestive Gland control
8 hr
.005
.002
trial i
.007
.003
8 hr
trial 2
mg NH,
-.004
+.004
+.072
+.090
+008
+.008
.000
+.004
+020
+.020
+.012
+.016
Bibliography
Albrecht (1920). J. Biol. Chem. 45, 395.
Baldwin and Needham (1934). Biochem. Jour. 28, 1373-1392.
Baldwin and Needham (1934), Biochem. Jour. 29, 238-261.
Baldwin, E. Dynamic Aspects of Biochemistry, fourth edition, Cambridge,
Cambridge University Press, 1963.
Florkin, M. Comparative Biochemistry: A Comprehensive Treatise,Ed.
Marcel Florkin and Howard S. Mason, New York, Academic Press,
1960.
Haden, R.L. (1923). J Biol. Chem. 56: 469-471.
Krebs and Henseleit (1932). Z. physiol. Chem. 210, 33.
McLeod, Onefrey, and Norris (1954). J. Bact. 68: 680-686.
Needham (1935). Biochem. Jour. 29, 238.
Sumner, J.B. (1926). J. Biol. Chem. 69: 435.