Int J Diabetes & Metabolism (2007) 15: 22-24
Catalase (antioxidant enzyme) activity in streptozotocin-induced diabetic rats
Durdi Qujeq,1 Timur Rezvani2
Department of Biochemistry and Department of Biophysics1, Faculty of Medicine2, Babol University of Medical Sciences,
Babol, Iran
____________________________________________________________________________________________________
Abstract
Background: High concentration and/or inadequate removal of reactive oxygen species may result in oxidative stress that
may cause severe metabolic malfunction. An imbalance in antioxidant enzymes has been related to specific pathologies such
as diabetic complications. Catalase catalyzes the reduction of hydroperoxides, thereby protecting mammalian cells against
oxidative damage. In addition, catalase is active in neutralizing reactive oxygen species and so removes cellular superoxide
and peroxides before they react with metal catalysts to form more reactive species. We investigated the status of catalase
activity in erythrocytes of streptozotocin (STZ)-induced diabetic rats. Method: Catalase activity was measured by using
spectrophtometric techniques. Result: Catalase activity increased in diabetic rats compared to control group [25.7 ± 2.8 vs.
16.3 ± 2.1 mmol H2O2 per min/ mg of protein, mean ± SD, p < 0.05]. Our results show that catalase activity increased
significantly in the erythrocytes of STZ-induced diabetic rats. (Int J Diabetes Metab 15: 22-24, 2007)
Key words: Antioxidant enzyme, Catalase, diabetic, oxygen free radicals.
described as oxidative stress. In some human diseases,
increased oxidative stress may make an important
contribution to disease pathology.8,9 ROS are generally
cytotoxic because of the oxidative damage they can cause to
cellular components. However, at low concentrations, ROS
may function as physiological mediators of cellular
responses.10 Oxidative stress, which is associated with the
formation of lipid peroxides, is suggested to contribute to
pathological processes in aging and many disease such as
diabetes, atherosclerosis and cataract.11 Increased oxidative
stress as a result of increased free radical formation has also
been suggested as a contributor to vascular damage in
diabetes.12-14 Low levels of ROS are indispensable in many
biochemical processes, including intracellular messaging in
cell differentiation and cell progression or the arrest of
growth, apoptosis,15 immunity16 and defense against
microorganisms.10,17 In contrast, high doses and/or
inadequate removal of ROS result in oxidative stress, which
may cause severe metabolic malfunction and damage to
biological macromolecules.18-20 Oxidative stress derived
from excessive superoxide production and an imbalance in
antioxidant enzymes has been related to many other
pathologies such as chronic granulomatous diseases,
diabetic complications, hepatitis, rheumatoid arthritis,
influenza virus, ulcer, pneumonia, HIV infection, cataract
and glaucoma.19,20 The aim of this study was to investigate
catalase activity in the erythrocytes of STZ -induced
diabetic rats.
Introduction
There are significant differences in the activities of
antioxidant enzymes between diabetic and non-diabetic
patients.1 The relationship between serum lipids,
lipoproteins, and the erythrocyte antioxidant enzymes,
catalase (CAT), glutathione peroxidase (GPx) and
superoxide dismutase (SOD) has been investigated in nonErythrocyte
insulin-dependent
diabetic
patients.2
antioxidant enzyme activities were measured in 105 noninsulin dependent diabetic patients, among whom 38 had
microvascular complications of diabetes.3 Free radicals and
lipid peroxide are easily formed in the diabetic state and
may play an important role in the development of diabetic
A
positive
correlation
between
complications.4
thiobarbituric acid reactive products and glucose, glycated
haemoglobin and fructosamine in the serum of diabetic
patients was observed.5 Impairment by streptozotocin (STZ)
of antioxidant enzymes may contribute to STZ-induced
experimental diabetes.6 SOD, GPx and catalase were
assayed in the erythrocytes of a diabetic population on
various treatment regimens in order to investigate any
relationships between their activities and diabetes markers.7
Reactive oxygen species (ROS) are constantly formed in the
human body and are removed by an antioxidant defense
system. In healthy individuals the generation of ROS
appears to be in approximate balance with antioxidant
defense. An imbalance between ROS and antioxidant
defenses in favour of the former has been
_____________________________________
Received on: 19/9/2006
Accepted on: 28/03/2007
Correspondence to Dr. Durdi Qujeq, Department of
Biochemistry and Biophysics, Faculty of Medicine , Babol
University of Medical Sciences, Babol, Iran, E-mail:
[email protected], Tel: +98-111-2229591-5(363)
Materials And Methods
Materials
2,4 -dinitrophenylhydrazine, H2O2, NaCl, EDTA,
trichloroacetic acid, HCl, ethanol-ethyl acetate, guanidine
hydrochloride and streptozotocin (STZ) were purchased
from Sigma (St. Louis, Mo, USA). Distilled water was used
in all experiments.
22
Activity(micro mole H2O2/min/mg of protein)
Catalase activity in diabetes
substrate solution (10 mM H2O2 prepared in 50 mM
phosphate buffer, pH 7.0) were compared with those of a
blank containing 1 ml phosphate buffer, instead of substrate
solution and 500μL of hemolysate dilution. The reaction
was initiated by the addition of the substrate solution and
incubated at 20 °C for about 1 min. Catalase activity was
expressed as mmol H2O2 /min/ mg protein. An enzyme unit
was defined as the amount of enzyme that catalyzes the
release of one μmol of H2O2 per min at 20 °C. Specific
activity was calculated in terms of units per mg of protein.
30
25
20
15
10
Results
All rats injected with STZ developed severe diabetes as
indicated by increasing serum glucose concentrations.
Serum glucose levels in diabetic rats were elevated
(approximately 2-fold) as compared with controls. Catalase
activity in the diabetic rat group was increased compared to
control group [25.7 ± 2.8 vs. 16.3 ± 2.1 mmol H2O2/ min/
mg of protein; mean ± SD. P<0.05] Catalase activities in
diabetic and control rats are reported in Figure 1.
5
0
1
2
Figure 1: Catalase activity in the erythrocytes of normal
and diabetic rats. Each column represents the mean value ±
SD of 6 separate experiments, p < 0.05. (column 1: diabetic
group; column 2: control group).
Discussion
Higher amounts of ROS have been shown to play a role in
the development of diabetic complications as well as in a
number of other disease states. As a safeguard against the
accumulation of ROS, intracellular enzymatic antioxidant
activities exist. ROS generated during metabolism can enter
into reactions that, when uncontrolled, can affect certain
processes leading to clinical manifestations.3,6,8 Oxidative
stress induced by excessive production of superoxide and
an imbalance in antioxidant enzymes has been linked to the
development of diabetic complications. ROS are key
participants in the damage caused by diabetic
complications. Diabetes is one of the pathological processes
known to be related to an unbalanced production of ROS,
such as hydroxyl radicals (HO), superoxide anions (O2) and
H2O2. Therefore, cells must be protected from this oxidative
injury by antioxidant enzymes. We found a significantly
higher catalase activity in diabetic rats compared with
controls. Our results confirm previous data indicating
enhanced ROS levels in diabetes mellitus.12,14 An
overproduction of ROS especially in diabetes can not be
properly balanced by antioxidant enzymes. Therefore, when
oxidative stress arises as a consequence of a pathologic
event, a defense system promotes the regulation and
expression of this enzyme. Our results indicate the presence
of some alteration in oxidant–antioxidant balance in the
erythrocytes of diabetic rats. The increase in the erythrocyte
antioxidant enzymes such as catalase is related to the
oxidative damage of membrane protein and lipid by
increased oxygen free radicals in the body.
Animals
Male rats (10 weeks old and between 220 and 260 g in body
weight) were randomly assigned to two groups. One group
of rats (diabetic group) received an intraperitoneal injection
of STZ (50 mg/kg) after anesthesia with diethyl ether. STZ
was dissolved in a citrate solution (0.15 M citric acid and
0.25 M sodium phosphate, pH 4.65). Another group
(control group) received an equivalent volume of citrate
buffer alone. Control and diabetic rats were caged
separately but housed under similar conditions. Both groups
of animals were fed with the same diet and water ad
libitum. All experimental manipulations were carried out
with the animals under diethyl ether anesthesia. On the day
of the experiments, blood samples were collected and
catalase activities were determined.
Preparation of blood samples and lysates
Blood was collected into heparinized syringes through
puncture of the left heart ventricle or tail. Erythrocytes
were obtained after centrifugation at 600-1400 x g for 10
min. Erythrocytes were washed twice with 0.9% sodium
chloride and were centrifuged under the same conditions. A
5% erythrocyte suspension in 0.15 M NaCl – 10 mM
sodium phosphate buffer, pH 7.4 was lysed by freezing (20°C) for 24 h and was used for catalase measurement.
Assay of catalase activity
Catalase (EC.1.11.1.6) catalyses the decomposition of
hydrogen peroxide to give water and molecular oxygen.
Catalase activity was determined according to a previously
reported method.21 The decomposition of H2O2 can be
followed directly by the decrease in absorbance at 240 nm.
The difference in absorbance per unit time is a measure of
catalase activity. The results obtained for the sample
containing 500 μL haemolysate dilution and 500μL
References
1. Jandric-Balen M, Bozikov V, Bistrovic D, et al.
Antioxidant enzymes activity in patients with
peripheral vascular disease, with and without presence
of diabetes mellitus. Coll Antropol 2003; 27: 735-743.
2. Kesavulu MM, Rao BK, Giri R, et al. Lipid
peroxidation and antioxidant enzyme status in Type 2
23
Durdi Qujeq & Timur Rezvani
diabetics with coronary heart disease. Diabetes Res
Clin Pract 2001; 53: 33-39.
3. Kesavulu MM, Giri R, Kameswara Rao B, et al. Lipid
peroxidation and antioxidant enzyme levels in type 2
diabetics with microvascular complications. Diabetes
Metab 2000; 26: 387-392.
4. Mano T, Shinohara R, Nagasaka A, et al. Scavenging
effect of nicorandil on free radicals and lipid peroxide
in streptozotocin-induced diabetic rats. Metabolism
2000; 49: 427-431.
5. Muchova J, Liptakova A, Orszaghova Z, et al.
Antioxidant systems in polymorphonuclear leucocytes
of Type 2 diabetes mellitus. Diabet Med 1999; 16: 7478.
6. Winkler R, Moser M. Alterations of antioxidant tissue
defense enzymes and related metabolic parameters in
streptozotocin-diabetic rats--effects of iodine treatment.
Wien Klin Wochenschr 1992; 104: 409-413.
7. Tho LL, Candlish JK, Thai AC. Correlates of diabetes
markers with erythrocytic enzymes decomposing
reactive oxygen species. Ann Clin Biochem 1988; 25:
426-431.
8. Halliwell B. The role of oxygen radicals in human
disease with particular reference to the vascular system.
Haemostasis 1993; 23: 118-126.
9. Gutteridge JMC. Lipid peroxidation and antioxidants
as biomarkers of tissue damage. Clin Chem 1995; 41:
1819-1828.
10. Bae YS, Kang SW, Seo MS, et al. Epidermal growth
factor induced generation of hydrogen peroxide. J Biol
Chem 1997; 272: 217-221.
11. Jianj ZY, Hunt JV, Wolff SP. Ferrous ion oxidation in
the presence of xylenol orange for detection of lipid
hydroperoxide in low density lipoprotein. Anal
Biochem 1992; 202: 384-389.
12. Gallou G, Ruelland A, Legras B, et al. Plasma
malondialdehyde in type 1 and type 2 diabetic patients.
Clin Chim Acta 1993; 214: 227-234.
13. Jennings PE, Jones AF, Florkowski CM, et al.
Increased diene conjugates in diabetic subjects with
microangiopathy. Diabet Med 1987; 4: 452-456.
14. Lyons TJ. Oxidized low density lipoproteins : a role in
the pathogenesis of atherosclerosis in diabetes? Diabet
Med 1991; 8: 411-419.
15. Ghosh J, Myers CE. Inhibition of arachidonate 5lipoxygenase triggers massive apoptosis in human
prostate cancer cells. Proc Natl Acad Sci 1998; 95:
13182-13187.
16. Yin GY, Yin YF, He XF. Effects of zhuchun pill on
immunity and endocrine function of elderly with
kidney -yang deficiency. Chung Kuo Chung His Chieh
Ho Tsa Chih. 1995; 15: 601-603.
17. Lee YJ, Galoforo SS, Berns CM. Glucose deprivation induced cytotoxicity and alterations in mitogenactivated protein kinase activation are mediated by
oxidative stress in multidrug-resistant human breast
carcinoma cells. J Biol Chem 1998; 273: 5294-5299.
18. Chopra S, Wallace HM. Induction of spermidine
/spermine N1-acetyltransferase in human cancer cells
in response to increased production of reactive oxygen
species. Biochem Pharmacol 1998; 55: 1119-1123.
19. Czene S, Tiback M, Harm S, Ringdahl M. PHdependent DNA cleavage in permeabilized human
fibroblasts. Biochem J 1997; 323: 337-341.
20. Wojtaszek P. Oxidative burst: an early plant response
to pathogen infection. Biochem J 1997; 322: 681-692.
21. Abei H. Catalse. Methods in Enzymatic analysis
(Bergmeyer HU,Ed),Verlag Chemie, Weinheim, 1974,
673-678.
24