C A R B O H Y D R A T E M E T A B O L I S M IN L I Z A R D S 1531

Studies on Carbohydrate Metabolism in Lizards

H . S . A L I A T H A R , S . N A Z R U L H A S N A I N , a n d M . ZAIN-UL-ABEDIN *

Department of Biochemistry, Univeristy of Karachi, Karachi-32, Pakistan

( Z . Naturforsch. 27 b , 1531—1535 [1972] ; received July 7, 1972)

Carbohydrate Metabolism, Lizards, Saccharoid Fraction

1. Initial glycogen levels in the liver were found to be significantly higher than in the kidneys
of the two species studied. The glycogen levels in kidney were however considerably higher than
in some mammalian species.
2. Total reducing capacity, true glucose, saccharoid fraction and titratable acidity increase
gradually with concomitant decrease in the glycogen levels when liver and kidney homogenates of
uromastix and liver homogenate of varanus were incubated for a period of 4 hours.
3. Total reducing capacity, true glucose and the titratable acidity increase gradually, while the
glycogen and the saccharoid fraction remain unchanged when the kidney homogenates of varanus
are incubated for 4 hours.
4. Total reducing capacity, true glucose and saccharoid fraction in the blood of uromastix are
higher than in varanus. The blood glucose levels in both the species are higher than found in the
mammals.

Carbohydrates are required by the living orga-
nisms to provide energy for work and other meta-
bolic activities. Metabolism of carbohydrates mainly
deals with the metabolism of glucose and closely
related substances. The principal route of carbo-
hydrate metabolism is through glycolysis and citric
acid cycle. Many workers have however contributed
in working out the intricate mechanisms of pentose
phosphate pathway1-4 and glucuronic acid
c y c l e 5 - 7 . The extent to which any one or more of
these pathways is used by any organism depends
upon several factors. Changes in the nature of
environmental conditions of the organism are as-
sociated with innumerable modifications in the pat-
tern of life. Many adaptive changes of structure and
behaviour are related to biochemical modifications
which have been associated with the environmental
changes. Reptiles occupy a pivotal position in the
animal kingdom because they serve as a link between
the poikilotherms and the homeotherms. Most of
the studies that have been conducted on carbo-
hydrate metabolism in reptiles are related to
seasonal variations in blood sugar levels 8 ' 9 , blood
lactic acid 9 and glycogen content of the tissues 10 .
The present study is concerned with the carbo-
hydrate metabolism 'in vitro' with particular re-
ference to blood sugar levels, saccharoid fraction of
Requests for reprints should be sent to H. S. ALI ATHAR,
Department of Biochemistry, University of Karachi,
Karachi-32, Pakistan.
* Present Address: Prof. M. ZAIN-UL-ABEDIN, Max-Planck-
Institut für experimentelle Medizin, D-3400 Göttingen,
W.-Germany.
blood, glycogen content of liver and kidney and
time course of changes in the above parameters and
titratable acidity of liver and kidney homogenates
of two reptilian species.
Materials and Methods
1. Animals: The lizards of local species of Uro-
mastix hardwickii and Varanus monitor were collected
from fields near Karachi. Animals of both sexes were
kept in wooden boxes without food for several days
before the experiments. The animals were collected
during the period May to October which is the active
period of these animals.
2. Preparation of tissue homogenates: Livers of
three animals were pooled and a 10% homogenate was
prepared in 0.02 M Tris-HCl buffer pH 7.36 + 0.1 M
NaCl, 0.0039 M KCl and 0.002 M CaCl 2 . In the case
of varanus kidneys of three animals and in case of
Uromastix kidneys of six to eight animals were homo-
genised in the above buffer to give a 5% homogenate.
3. Studies with tissue homogenates: Samples of liver
and kidney homogenates were drawn immediately at
zero time and analysed for initial glycogen, glucose,
total reducing capacity and titratable acidity. The rest
of homogenates were incubated at 37 °C in a water bath
with frequent shaking. Samples were drawn after 30,
90, 180 and 240 min and analysed for above para-
meters.
a) Glycogen in the homogenates was precipitated
with ethanol11 and then determined according to the
method of F A L E S 1 2 .
b) True glucose was determined by N E L S O N - S O M O -
GYI method 13.
c) Total reducing capacity of the homogenates at
various time intervals was determined by F O L I N - W U
method 14.

Unauthenticated
Download Date | 6/19/19 2:41 PM
1532 H. S. A L I A T H A R , S. N. HASNAIN, A N D M. ZAIN-UL-ABEDIN

d) Titratable acidity: 5 ml of the homogenates at
each time intervals was transferred to a centrifuge tube
and immediately immersed in a boiling water bath for
5 min, cooled and centrifuged. A 2 ml aliquot was
titrated against 0.02 M NaOH, using 2 drops of thymol
blue as indicator.
4. Initial glycogen content of the tissues: Liver and
kidney of the animals were removed immediately after
sacrifice and rinsed in chilled saline. A piece of each
tissue was transferred to a tube containing warm KOH.
Glycogen was precipitated with ethanol and estimated
by anthrone method.
The difference between the values of total reducing
capacity and true glucose was taken as saccharoid
fraction. Total reducing capacity and true glucose were
also determined in blood of the two species.
Results
The initial glycogen levels in the liver and kid-
ney of uromastix and varanus are shown in Table I.
The initial glycogen levels were found to be 3.96
Table I. Glycogen content of liver and kidney of uromastix
and varanus. The values are expressed as mg glucose per 100
mg wet wt. tissue.
Animal Liver Kidney
Uromastix 3.96 4- 0.48 * 0.33 ± 0.01
(8) (10)
Varanus 4.62 ± 0.47 0.23 ± 0.01
(11) (12)
* Numbers in parentheses indicate the number of observations.
Each value represents a mean + S.E.
and 0.33 mg-% in the liver and kidney of uromastix
respectively, whereas the liver and kidney of varanus
were found to contain 4.62 and 0.23 mg-% glycogen
respectively. Thus the glycogen concentration in the
liver of uromastix was 10 times that of its kidney
while in varanus the glycogen content in liver was
20 times more than in kidney.
Table II shows the time course of changes in
carbohydrate content and titratable acidity in the
liver homogenate of uromastix. The total reducing
capacity, true glucose, saccharoid fraction and
titratable acidity increased gradually whereas glyco-
gen content decreased. The total reducing capacity
and true glucose levels increased from 0.38 to
1.23 mg-% and 0.19 to 0.66 mg-% respectively in
4 hours. The saccharoid fraction was increased 2§
times, from 0.2 to 0.57 mg-%. The titratable aci-
dity increased from 13.9 to 17.0 //eq/100 mg tissue.
The glycogen levels were however decreased from
1.97 to 0.5 mg-%.
The time course of changes in carbohydrate
content and titratable acidity in the kidney homo-
genate of uromastix is shown in Table III. The true
glucose and total reducing capacity are more than
doubled in 4 hours. The total reducing capacity was
increased from 0.39 to 0.82 mg-%, whereas true
glucose increased from 0.18 to 0.45 mg-%. The sac-
charoid fraction was increased from 0.21 to 0.40
mg-%, similarly titratable acidity increased from
12 .75 to 14.2 /ceq/100 mg tissue. During this period
of time the glycogen level fell from 0.17 to 0.06
mg-% hence it was reduced to about J in 4 hours.
The time course of changes in carbohydrate con-
tent and titratable acidity in the liver homogenate
of varanus is shown in Table IV. The total reducing
capacity and true glucose was increased 3 to 4 folds
in 4 hours. The total reducing capacity was in-
creased from 0.67 to 1.87 mg-% whereas true glu-
cose level was increased from 0.28 to 1.17 mg-%.
The saccharoid fraction and the titratable acidity
also increased from 0.18 to 0.45 mg-%. The
saccharoid fraction was increased from 0.39 to
0.70 mg-%. The titratable acidity was increased
from 12.1 to 16.0/<eq/l00 mg tissue, hence an in-
crease of 32% in 4 hours. The glycogen level
registered a sharp decline in the first 90 min (from

Table II. Time course of changes in carbohydrate content and titratable acidity in the liver homogenate of uromastix.

Time Carbohydrates mg glucose/100 m g tissue wet weight Titratable A c i d i t y

[min] Total R e d u c i n g True Glucose Saccharoid Glycogen (/<eq o f lactate/100 m<
Capacity Fraction tissue)

0 0.38 ± 0.02 * 0.19 ± 0.03 0.20 ± 0.02 1.97 ± 0.55 13.9 ± 0.89
30 0.68 ±0.10 0.32 ±0.08 0.36 ± 0.02 1.39 ± 0.46 15.4 ± 0.87
90 0.91 ±0.13 0.54 ± 0.13 0.37 ± 0.03 0.91 ± 0.32 16.4 ± 0.90
180 1.07 ±0.13 0.59 ± 0.08 0.48 ±0.11 0.69 ± 0.20 16.4 ± 0.65
240 1.23 ±0.15 0.66 ±0.01 0.57 ± 0.15 0.50 ± 0.15 17.0 ± 0.49

* Mean + S.E. Each value is a mean of 4 observations.

Unauthenticated
Download Date | 6/19/19 2:41 PM
 C A R B O H Y D R A T E M E T A B O L I S M IN L I Z A R D S 1533

Table III. Time course of changes in carbohydrate content and titratable acidity in the kidney homogenate of uromastix.

Time Carbohydrates m g glucose/100 m g tissue wet weight Titratable A c i d i t y

[min] Total R e d u c i n g True Glucose Saccharoid Glycogen (//eq o f lactate/100 m g
Capacity Fraction tissue)

0 0.39 ± 0.01 * 0.18 ±0.01 0.21 ± 0.02 0.17 ± 0.01 12.75 ± 1.65
30 0.55 ± 0.02 0.25 ± 0.01 0.30 ± 0.005 0.10 ± 0.01 13.15 ± 1.52
90 0.68 ± 0.01 0.35 ± 0.02 0.33 ± 0.08 0.08 ± 0.02 13.50 ± 1.57
180 0.78 ± 0.01 0.39 ± 0.03 0.39 ± 0.08 0.08 ± 0.01 13.85 ± 1.57
240 0.82 ± 0.01 0.45 ± 0.05 0.40 ± 0.06 0.06 4- 0.01 14.20 ± 1.46

* Mean + S.E. Each value is a mean of 4 observations.

Table IV. Time course of changes in carbohydrate content and titratable acidity in the liver homogenate of varanus.

Time Carbohydrate mg glucose/100 m g tissue wet weight Titratable A c i d i t y

[min] Total R e d u c i n g True Glucose Saccharoid Glycogen (//eq o f lactate/100 m g
Capacity Fraction tissue)

0 0.67 ± 0.04 * 0.28 ± 0.01 0.39 ± 0.03 3.40 ± 0.50 12.10 ± 0.36
30 1.10 ± 0.06 0.58 ± 0.01 0.52 ± 0.05 2.40 ± 0.65 12.95 ± 0.28
90 1.43 ±0.07 0.99 ± 0.06 0.51 ± 0.02 0.84 ± 0.23 14.75 ± 0.62
180 1.79 ± 0.12 1.12 ± 0.10 0.67 ± 0.07 0.45 ± 0.12 16.0 ±0.44
240 1.87 ± 0.12 1.17 ± 0.08 0.70 ± 0.09 0.30 ± 0.02 16.0 ±0.5

* Mean ± S.E. Each value is a mean of 4 observations.

Table V. Time course of changes in carbohydrate content and titratable acidity in the kidney homogenate of varanus.

Time Carbohydrate mg/100 mg tissue wet weight Titratable Acidity

[min] Total R e d u c i n g True Glucose Saccharoid Glycogen (//eq lactate/100 mg
Capacity Fraction tissue)

0 0.43 ± 0.08 * 0.10 ± 0.01 0.33 ± 0.06 0.037 ± 0.01 10.30 ± 0.79
30 0.40 ± 0.03 0.14 ± 0.02 0.26 ± 0.04 0.035 ± 0.01 11.35 ±0.72
90 0.52 ± 0.06 0.18 ± 0.03 0.34 ± 0.04 0.061 ± 0.01 12.15 ± 1.08
240 0.61 ± 0.06 0.25 ± 0.03 0.36 4 - 0.03 0.037 ± 0.01 12.30 ± 1.04

* Mean ± S.E. Each value is a mean of 4 observations.

3.40 to 0.84 mg-%) and was then reduced gradually Table VI. Saccharoid fraction of blood of uromastix and
varanus.
to 0.30 mg-% at the end of 4 hours incubation
period. During the total incubation period the glyco- Animal Total True Saccharoid
gen level was reduced to l / l 0 the initial levels. Reducing Glucose Fraction
Capacity
Table V represents the time course of changes in
mg-% mg-% mg-%
carbohydrate content and titratable acidity in the
kidney homogenate of varanus. The total reducing Uromastix 157.80 ± 3 . 1 0 * 122.11 ± 4.91 35.74 ± 3 . 1 2
capacity and true glucose levels increased gradually (10) (10) (10)
Varanus 138.30 ± 9.65 107.30 ± 7.20 31.0 ± 3 . 6
from 0.43 to 0.61 mg-% and 0.10 to 0.25 mg-% (6) (6) (6)
respectively. The saccharoid fraction however did
not increase significantly. The titratable acidity was * Mean + S.E. Numbers in parentheses indicates the number
of observations.
increased from 10.3 to 12.3//eq/100 mg tissue. The
glycogen level remained constant during 4 hour
Discussion
period of incubation.
The saccharoid fraction in blood of uromastix The two reptilian species used in the present stu-
and varanus is shown in Table VI. The saccharoid dies — namely uromastix and varanus are taxonomi-
fraction in blood of uromastix and varanus were cally very closely related to each other. Both of
approximately 35.74 and 31.0 mg-% respectively. them belong to the natural order Lacertilia and have

Unauthenticated
Download Date | 6/19/19 2:41 PM
1534 H. S. A L I A T H A R , S. N. H A S N A I N , A N D M. ZAIN-UL-ABEDIN
adapted for the desert life and live deep in burrows
in sandy areas. Inspite of this they do have anatomi-
cal and physiological differences distinct enough to
place them in two different genera.
The glycogen levels in the kidney and liver tis-
sues (Table I) are very much similar in both the
species. The livers of uromastix and varanus were
found to contain 3.96 and 4.62 mg glycogen per
100 mg wet wt of tissue. These values are similar
to those observed in other animal species. W E B E R
and CANTERO 15 reported 4 mg-% glycogen in rat
liver while L Y O N and P O R T E R 16 reported 5 . 0 7 mg-%
in mice liver. In human beings the liver glycogen
content is 3.15 mg-% 17 while in a lizard Varanus
greseus, it is 4.4 mg-% 10 . The glycogen content in
kidneys of both the reptiles is considerably less in
comparison to that of their livers. This is not sur-
prising because liver plays an important role in ac-
cumulating and storing the excess sugar from the
blood in the form of tissue glycogen. This reserve
carbohydrate is released into the blood circulation
when sugar level falls below a critical level in the
blood. Such a regulatory mechanism by the liver
has been known for a long time.
Although the levels of liver glycogen in various
species are about the same, there is a considerable
difference in the glycogen levels of the kidneys of
different species. KREBS et al.18 have reported 0 . 0 1
mg-% glycogen in rat kidney but higher glycogen
content in the kidneys of reptiles has been re-
ported 19. Perhaps the high glycogen content in the
kidneys of these reptiles indicates a higher meta-
bolic activity in this tissue as compared to mammals.
During the course of incubation of the liver
homogenate of uromastix (Table II) over a period
of 4 hours there was a gradual drop in the glycogen
content while the total reducing capacity, true glu-
cose, saccharoid fraction and titratable acidity in-
creased concomitantly. During the process of gly-
colysis when glycogen breaks down, pyruvate and
lactate will be accumulated, alongwith an increase in
the free glucose and sugar phosphate levels. The
total reducing capacity usually includes besides the
1 E. RACKER, Advances in Enzymol. 15, 141 [1954].
2 E. RACKER, Harvey Lectures 5 1 , 1 4 3 [1955].
3 B . L . HORECKER a n d A . H . MEHLER, A n n . R e v .
2 4 , 2 0 7 [1955].
4 H. G. WOOD, Physiol. Rev. 35, 841 [1955].
5 S. HOLLMANN a n d 0 . TOUSTER, N o n - G l y c o l y t i c
of Metabolism of Glucose. Academic Press, New York
1964.
true glucose a number of other components in-
cluding sugar phosphates 20 . The difference between
the total reducing capacity and true glucose is re-
ferred to as saccharoid fraction. During the break-
down of glycogen the increase in saccharoid fraction
indicates the accumulation of sugar phosphates.
Since the end product of glycolysis is the pyruvic
and/or lactic acid, the increase in titratable acidity
therefore is not surprising.
Similar pattern of changes in total reducing capa-
city, true glucose, saccharoid fraction, titratable aci-
dity and glycogen was observed in the liver homo-
genate of varanus (Table IV).
The time course of changes in carbohydrate con-
tent and titratable acidity in the kidney homogenate
of uromastix (Table III) show that total reducing
capacity, true glucose, saccharoid fraction and
titratable acidity increased gradually with time
whereas glycogen decreased. Time course of changes
in total reducing capacity, true glucose, saccharoid
fraction, titratable acidity and glycogen in the kid-
ney homogenate of varanus is shown in Table V. A
gradual increase in total reducing capacity, true
glucose and titratable acidity was noted whereas the
saccharoid fraction and glycogen content showed no
significant change.
Kidney homogenates of uromastix and varanus
showed a different pattern of changes. Increase in
total reducing capacity, true glucose, and saccharoid
fraction was more in case of uromastix than in
varanus and specifically saccharoid fraction showed
no signi cant change in the latter.
The total reducing capacity, true glucose and the
saccharoid fraction were higher in the blood of
uromastix than in varanus (Table V I ) . These levels
are however significantly higher than those found in
mammals 21 . The saccharoid fraction is related to the
blood sugar level and varies with total reducing
capacity. This is in agreement with the findings of
K H A N and R A H M A N 22 who reported that the change
in the value of saccharoid fraction was similar to
that of sugar and the behaviour was similar in nor-
mal as well as in diabetes.
6 J . J . BURNS, A m e r . J . M e d . 2 6 , 7 4 0 [1959].
7 J. L. STROMINGER. Physiol. Rev. 40, 55 [ I 9 6 0 ] .
Biochem. 8 M . ZAIN-UL-ABEDIN a n d M . H . QAZI, C a n a d . J. Biochem.
43, 831 [1965].
9 E. HUTTON and J. GOODNIGHT, Physiol. Zool. 30, 198
Pathways [1957].
10 G . HAGGAG. K . A . RAHIM, and F. KHALIL, C o m p . Biochem.
Physiol. 16, 457 [1965],
Unauthenticated
Download Date | 6/19/19 2:41 PM
 SODIUM SUBSTITUTES A N D R E C E P T O R POTENTIAL 1535

11 HASSID and ABRAHAM, Chemical Procedures for Analysis 17 D. S. MACINTYRE, S. PEDERSEN, and W. G. MADDOCK,
of Polysaccharides, i n : Methods of Enzymology, V o l . 3, Proc. Soc. exp. Biol. Med. 47, 354 [1941].
Academic Press, New York 1957. 18 H. A. KREBS, D. A. H. BENNETT, P. DE GASQUET, T.
12 F. W. FALES, J. biol. Chemistry 193, 113 [ 1 9 5 1 ] . GASCOYNE, a n d T . YOSHIDA, B i o c h e m . J. 8 6 , 2 2 [1963].
13 N. NELSON and M. SOMOGYI, as cited in Practical clinical 19 M. ZAIN-UL-ABEDIN and B. KATORSKI, Biochem. J. 102,
Biochemistry, H. VARLEY, 4th edition, W. HEINEMANN, 189 [1967].
Medical Book Ltd., London 1967. 20 C. N. GRAYMORE a n d M. J. TOWLSON, Nature [London]
14 O. FOLIN and H. W u , as cited by OSER, in Hawk's Practical 195, 76 [1962].
Physiological Chemistry, 14th edition, McGraw-Hill, New 21 J. S. ANINO, Clinical Chemistry, Principles and Procedures,
York 1965. 3rd ed. Boston, Little-Brown 1954.
15 G. WEBER and A. CANTERO, Science [Washington] 120, 22 I. A . KHAN and M. A. RAHMAN, Nature [London] 215,
851 [1954]. 979 [1967].
16 J. B. LYON and J. PORTER. J. biol. Chemistry 238, 1
[1963].

The Effect of some Sodium Substitutes on the Receptor Potential

of the Crayfish Photoreceptor Cell
H . STIEVE, H . GAUBE, a n d T . MALINOWSKA

Institut für Neurobiologie der KFA Jülich

(Z. Naturforsch. 27 b , 1535—1546 [1972] ; received September 14, 1972)

Receptor potential, Na + -substitutes, ion dependence, photoreceptor cell, Crustacea

Isolated crayfish retinas were perfused with four solutions in which Li + , NH 4 + , Tris H + and
glucose were substituted for the sodium ions in the physiological salt solution.
The changes of the extracellularly recorded receptor potential (ReP) evoked by short or long
stimuli were measured. The changes in the shape of ReP by test solutions were different for each
Na-substitute.
For lithium ions as a Na-substitute (Tab. I and Fig. 3) the plateau value he was considerably
decreased (to ~ 2 0 % ) contrary to the peak-amplitude /i m ax which even slightly increased.
Ammonium ions show quite a different effect than all the other substitutes. The ReP is de-
creased strongly and irreversibly (Tab. II and Fig. 4 ) .
When Tris (hydroxymethyl-ammoniummethane-hydrochloride) is substituted for Na, Amax de-
creased to about 60 per cent and the plateau is even more reduced (to 20 per cent; Tab. III). Only
the recovery-value for he (50% smaller) is markedly different contrary to our former experiments
where choline was used as Na-substitute (decrease 20%).
Glucose as a substitute for sodium chloride caused strongly decreased peak-amplitude Amax 26%
(Tab. IV and Fig. 9 ) . Increased osmotic pressure due to excess glucose causes irreversible damage
of the R e P (Tab. V ) . All the changes except those produced by NH 4 + were reasonably reversible.
The results can be explained by the following assumptions:
a) the maximum of the ReP is caused mainly by an increase in the permeability of the cell mem-
brane for sodium. Ca- and Mg-ions also contribute to it to a certain degree.
b) The plateau value of the R e P to long light stimuli is determined:
1. by the sodium concentration gradient,
2. by active transport processes,
3. by the Ca ++ - and Mg^-gradients,
4. the chloride gradient may perhaps contribute to this value.

It is generally accepted that the receptor potential
of the invertebrate retina is caused by ion currents
across the visual cell membrane. In this process
positive ions permeating from the external medium
into the cell play a decisive role.
In earlier publications we have reported ion sub-
stitution experiments in the crustacean retina
Requests for reprints should be sent to Prof. Dr. H. STIEVE,
Institut für Neurobiologie d. KFA Jülich GmbH., D-5170
Jülich 1, Postfach 365.
(STIEVE 1 ' 2 ). When external sodium was replaced
by choline, we observed effects which were at least
in part a specific result of the substitute ion. The
present paper deals with a series of experiments per-
formed with different sodium substitute ions in order
to determine more exactly the specific role of sodium
in the process leading to the receptor potential.
One should keep in mind that sodium ions play a
different role in the visual system of invertebrates as
compared to vertebrates; and that they possibly per-

Unauthenticated
Download Date | 6/19/19 2:41 PM