Full research publication
Registration number: 15-43-8-42
Topic section: Biochemical research
Subsection: Bioorganic chemistry
The article is published as a material of distance participation in the International scientific forum
‘Butlerov’s Heritage-2015’. http://foundation.butlerov.com/bh-2015/
UDC 577.15: 557.15: 616.72. Received for publishing on 26th April 2015
The state of antioxidant enzymes system
of persons with diabetes mellitus type II
© N.V. Kirillova*+1, M.G. Mescheryakova1, O.M. Spasenkova1, I.A. Orehova1, V.I. Laryonova2,
A.N.Voytovych2
1
Department of biochemistry. Saint-Petersburg State Chemical-pharmaceutical academy. Prof. Popova str.,14.
Saint-Petersburg 1977376. Russia
2
Federal State Budgetary Scientific Institution “Institute of Experimental Medicine”, Akad. Pavlov
str., 12. Saint-Petersburg 1977376. Russia
Тел.: +7(812) 234-90-33. Факс: 234-60-44. Е-mail: [email protected]
[email protected]
Key words: diabetes mellitus, SOD, catalase, glutathione peroxidase, glutathione reductase,
paraoxonase
Abstract
It was revealed that the activity of key antioxidant enzymes in blood of patients with diabetes mellitus
type II: SOD, catalase, glutathione peroxidase, glutathione reductase and paraoxonase is decreasing. It was
found that oxidative stress in patients with diabetes mellitus type II is increasing.
Introduction
Diabetes is not only a risk factor for many cardiovascular diseases, but is often the cause of
early mortality [1, 2]. Currently, the number of diabetes cases in the world is constantly growing and
it is likely that in 20 years the number of patients with this pathology will increase by 120%,
moreover men have greater risk of having diabetes compared with women (the risk is 2:3 and 3:5,
respectively) [3]. In addition, life expectancy of at least 60% of patients with diabetes mellitus type II
reduces in connection with the development of coronary heart disease and its complications [4, 5]. It
is known that 8 % of the US population aged over 20 years is suffering of diabetes, 12% - aged 40 to
74 years, and 19% older than 75 years. Moreover, about 35% of the surveyed patients did not know
that they have diabetes before the detection of elevated levels of blood glucose fasting. Similar data
was obtained in the study of populations of other advanced countries [6]
It is now established [7-10] that increased production and high content of superoxide radical
was observed in the serum of patients with diabetes mellitus, therefore, one of possible ways to
reduce the biosynthesis of insulin is the aconitase inhibition by active forms of oxygen. Aconitase is
one of the enzymes of the citric acid cycle and is the main supplier of high energy substances in cells.
In addition, the high level of prooxidants may directly participate in the damage of β-cells of the
pancreas. Also, revealed hyperglycemia is the cause of the whole organism's systemic reactions.
Antioxidant enzymes system plays a major role in protecting cells from oxygen radicals. Antioxidant
enzymes can neutralize superoxide radicals and peroxide compounds, for example, superoxide
dismutase (SOD) damages superoxide radicals. Catalase, peroxidase, glutathione peroxidase and
paraoxonase inactivate hydrogen peroxide. Paraoxonase is an enzyme with antioxidant properties,
which is associated with the HDL and protects them from the effects of oxygen radicals [11, 12] in
plasma.
In this work we studied the activity of key antioxidant enzymes: SOD, catalase, paraoxonase,
glutathione peroxidase and glutathione reductase.
Experimental part
In this investigation, 180 men and 95 women aged 40 to 60 years with a diagnosis of diabetes N type
were examined. The control group consisted of 60 persons of the same age (37 men and 23 women).
In both groups blood sampling was performed in the morning fasting, heparin was an anticoagulant.
42____________© Butlerov Communications. 2015. Т.43. №8. _________ Kazan. The Republic Of Tatarstan. Russia.
THE STATE OF ANTIOXIDANT ENZYMES SYSTEM OF PATIENTS…__________________________________42-46
The state of the antioxidant enzymes system was evaluated by the methods of spectrophotometry, the
activities of SOD, catalase, glutathione peroxidase, glutathione reductase and paraoxonase were
estimated. SOD activity was determined by using the reaction of superoxide-dependent oxidation of
quercetin [13]. Catalase activity was estimated by the rate of decomposition of hydrogen peroxide
[14]. Phenylacetate was a substrate for determination of arylesterase paraoxonase activity [15, 16].
The activities of glutathione peroxidase and glutathione reductase were determined according to the
methods [17, 18]. Statistical processing of results was performed using the program Stanistica 5.0
Windows.
Results and discussion
SOD is the key enzyme of the tissues antioxidant system. SOD limits the processes of
conversion of superoxide radical in other active forms of oxygen, as it catalyzes the reaction of
formation of hydrogen peroxide and triplet oxygen from superoxide anion radical. SOD is a critical
component of the antioxidant enzymes system, as superoxide radicals generated in the cells can react
with hydrogen peroxide with the formation of more reactive short-living oxygen radicals. These
radicals are the greater risk to the life of the cells. In this regard, the higher the level of SOD activity,
the more intensively superoxide radicals will be involved in the reaction of dismutation and the less
these dangerous short-living radicals will be produced [19].
Table 1. SOD and catalase activity in the blood during diabetes mellitus type II
Control group
Group with diabetes mellitus type II
Catalase content in erythrocyte suspension, units/mg Hb
n=14
0.03±0.03
p<0.05
6.18 + 0.87
n=10
0.38±0.36
p<0.05
n=25
n=8
2.85±1.20
p<0.05
SOD content in total blood, units (%)
n=9
0.45±0.34
p<0.05
52.40+4.46
n=15
2.26±0.68
p<0.05
n=37
n=7
6.13±1.19
p<0.05
n=9
24.98±3.26
p<0.05
n=3
62.10±0.21
р≤0.05
This investigation has shown, that SOD activity was reduced in blood of 91% of patients with
diabetes mellitus, and trace amounts of SOD were found in blood of 21% of patients from this group.
7% of patients оnly with diabetes mellitus demonstrated elevated levels of SOD activity in the blood.
It was comparably or even slightly higher than the activity of the enzyme in the healthy people's
blood (see table 1).
The characteristic function of catalase is highly efficient catalysis of disproportionation
reaction of hydrogen peroxide molecules that resulting in formation of water and molecular oxygen.
It is also known that the content of catalase in the aerobic cells is closely linked to the metabolism of
oxygen active forms. These active forms are one of the elements of the signals cascade that lead to
run of cells adaptive mechanisms, including changing the speed of antioxidant enzymes synthesis
[20]. The more catalase activity is found in the cell, the lower is the degree of oxidative damage of
proteins [21]. Moreover, superexpression of catalase in the aerobic cells is able to increase vitality
and to reduce apoptosis in cells treated by lipoperoxide in vitro [22]. As shown by our study, catalase
activity was significantly reduced in all cases of diabetes mellitus type II (see table 1).
Glutathione peroxidase - selenium containing enzyme - inactivates hydrogen peroxide in the
cells of higher animals.
© Butlerov Communications. 2015. Т.43. №8. _________
E-mail: [email protected] _______________ 43
Full research publication ________________ N.V. Kirillova, M.G. Mescheryakova, O.M. Spasenkova, I.A. Orehova,
V.I. Laryonova & A.N.Voytovych
Table 2.Glutathione peroxidase and glutathione reductase activity
in the blood during diabetes mellitus type II
Control group
Group with diabetes mellitus type II
Glutathione peroxidase in erythrocyte suspension, mol . 10-6/min per 1g of Hb
n=4
1.13±0.67
p<0.05
n=8
25.42±3.18
р0.05
19.57+3.21
n=18
40.13±2.80
p<0.05
n=37
n=29
45.54±4.33
p<0.05
n=16
55.08±2.71
p<0.05
n=11
64.07±4.09
p<0.05
The activity of glutathione reductase, mol/min per 1 g of Hb
n=10
traces
p<0.05
n=10
0.53±0.24
p<0.05
n=8
1.19±0.23
p<0.05
11.10+2.27
n=9
2.19±0.18
p<0.05
n=37
n=18
3.08±0.30
p<0.05
n=14
3.9±0.31
p<0.05
n=10
5.80±0.90
p<0.05
n=5
9.89±1.05
р0.05
n=2
14.15±0.57
р0.05
n=5
19.98±2.24
р≤0.05
Glutathione peroxidase catalyzes the reaction of unstable organic hydroperoxides reduction by
glutathione, including hydroperoxides reduction of polyunsaturated fatty acids, stable substances –
hydroxyacids. Аffinity of glutathione peroxidase to hydrogen peroxide is higher than that of catalase,
so the first one works more effective at low concentrations of hydrogen peroxide. At the same time,
catalase plays the key role in cells protection from oxidative stress caused by high concentrations of
H2O2. Two molecules of glutathione are oxidized and connected by disulfide linkage that resulting in
formation of oxidized glutathione in many reactions catalyzed by glutathione peroxidase. Then
special enzyme – glutathione reductase – restores oxidized glutathione in the cells. In this work it was
found that glutathione peroxidase activity in the blood of 4-5% of persons with diabetes mellitus was
reduced, enzyme activity of 9-10% of patients was found in limits of reference values and compared
with the rate for healthy people enzyme activity of 86% of patients had increased. Glutathione
reductase activity in the blood of 87% of diabetic patients was decreased (moreover, for 21-22% of
patients, up to trace quantities), about 8% of patients had enzyme activity in limits of reference
values, and enzyme activity of 5.5% of patients was higher than reference values (see table 2).
In recent years, the enzyme – paraoxonase – is the subject of great attention of the researchers.
It is esterase calcium dependent, which is fully associated with HDLP. High density lipoproteins as
known are protecting from LDL oxidation. It is due to the presence of antioxidant enzymes that
hydrolyze lipoperoxide in oxidized low-density lipoproteins. Paraoxonase is the most important
antioxidant enzyme [11, 12].
Determination of paraoxonase activity in blood serum had shown that enzyme activity
significantly differed compared with reference values for 80% of patients under research (see table
3). In this case, enzyme activity in plasma of 67% of patients was dramatically reduced, and 11% of
patients had values increased significantly.
Table 3. Paraoxonase activity in the blood during diabetes mellitus type II
Control group
Group with diabetes mellitus type II
The activity of the enzyme in plasma, mol . 10-6/min . ml
n=4
1.78±0.31
p<0.05
22.67+2.96
n=6
6.84±1.02
p<0.05
n=25
n=14
13.53±0.64
p<0.05
n=8
20.87±2.98
р0.05
n=4
32.38±3.10
p<0.05
44 _______ http://butlerov.com/ _______ ©Butlerov Communications. 2015. Vol.43. No.8. P.42-46. (English Preprint)
THE STATE OF ANTIOXIDANT ENZYMES SYSTEM OF PATIENTS…__________________________________42-46
Conclusion
Formation of oxygen active forms continuously occurs in aerobic cells. It is a significant
potential hazard and the pathogenetic role of prooxidants for a large number of human diseases is
shown to date. Oxidative stress is a characteristic for most diseases, which compounded by impact of
oxygen radicals. In turn, it is accompanied by activation of free radical processes in cells [19, 23 25]. Therefore, normal physiological functioning and development of cells requires the following
condition: oxygen active forms production which toxic for cells must constantly be balanced by their
inactivation by antioxidant protective system.
Analyzing our results, we may conclude that persons with diabetes mellitus type II have
dramatically reduced enzymatic antioxidant potential in comparison to healthy people. Intensification
of oxidative stress takes place due to prooxidants accumulation.
Summary
1. Аctivity of key antioxidant enzymes was reduced for persons with diabetes mellitus type II.
2. Timely detection of low antioxidative activity in the blood will adjust and optimize the medical
treatment. It is important for primary prevention of this disease.
References
[1]. Aleksandrov A. A. Diabetes mellitus ischemic heart disease: an unsolved mystery of sulfonamides. //
Cons-med. 2001. V. 10, P. 1-6.
[2]. Aleksandrov A. A. Antidiabetic therapy and heart: the burden of proof. // Cons-med. 2002. V. 4(10), P. 39.
[3]. Aleksandrov A. A. Diabetes mellitus: the disease "exploding plaques". // Cons-med. 2001. V. 10, P. 12-19.
[4]. Karpov Y. A. Arterial hypertension when diabetes mellitus: the main directions of treatment. // RMJ.
2001. V. 9(24), P. 2-9.
[5]. Hamdan A.D., Quist W.C., Gagne J.B. et al. Angiotensin-converting enzyme inhibition suppresses
plasminogen activator inhibitor-1 expression in the neointima of balloon-injured rat aorta // Circulation. 1996.
V. 93, P.1073-1078
[6]. Elagin R. I. Profitability of hyperlipidemia treatment for patients with diabetes mellitus. // Cons-med.
2001. V. 3(2), P. 11-17.
[7]. Gatshko G. G., Mashul L. M., Sablinskaya O. V. Age peculiarities of lipid peroxidation in the blood
during alloxan diabetes. // Probl. endocrinol. 1985. No. 2, P. 68-70.
[8]. Zanozin O. V. Role of oxidative stress in the development and progression of late complications of
diabetes mellitus type 2. The possibility of antioxidant therapy. // Avtoref.diss.doctora med.nauk. Nizhny
Novgorod, 2010.
[9]. Kozlov G.S., Balabolkin M. I., Buidina T. A. Possibility of insulin correction of some metabolic changes
during insulin-dependent diabetes mellitus. // In the book: Surgical diseases and diabetes. M., 1989, P. 53-56.
[10]. Nikolaeva, L. P., Cherdantsev D. V., Stepanenko A.V., Dyatlov V. Y. Change of the state of the
antioxidant system of patients with diabetic foot syndrome. // Fundamental research. 2010. No. 11, P. 95-97.
[11]. Draganov D.I, La Du B.N. Pharmacogenetics of paraoxonases// J. Lipid Research. 2000. V.41, P. 13581363.
[12]. Takeshi Kujiraoka, Tomoichiro Oka, Mitsuaki Ishihara, Tohru Egashira and et. A sandwich enzymeliked immunosorbent assay for human serum paraoxanase concentration // J. Lipid Reaseach. 2000. V.41, P.
1358-1363.
[13]. Kostyuk V. A., Potapovich A. I., Kovaleva J. V. Simple and sensitive method for the determination of
superoxide dismutase activity based on the oxidation of quercetin // Vopr. med. chimii. 1990. Vol. 36. No. 2,
P. 88-913.
[14]. Beutler E. Red cell metabolism а manual biochemical methods // New York: Grune Stratton. 1975. Р.8990.
[15]. Suda N., Morita I., Kuroda T., Murota S. Parcipitation of oxidative stress in the process of osteoclast
differentiation // Biochim. Biophys. Acta. 1993. V.115, №3, P.318-323.
[16]. Kujiraoka T., Oka T., Ishihara I., Egashira T., Fujioka T., Saito E., Saito S., Miller N.E., Hattori H. A
sandwich enzyme-linked immunosorbent assay for human serum paraoxonase concentration // J. Lipid
Research. 2000. V. 41. P. 1358-1363.
[17]. Moin V. I. Simple and sensitive method for the determination of glutathione peroxidase in erythrocytes
// Lab. delo. 1986. No. 12, P. 724-727.
© Butlerov Communications. 2015. Т.43. №8. _________
E-mail: [email protected] _______________ 45
Full research publication ________________ N.V. Kirillova, M.G. Mescheryakova, O.M. Spasenkova, I.A. Orehova,
V.I. Laryonova & A.N.Voytovych
[18]. Rao M.V., Paliyath G., Ormrod D.P. Ultraviolet-B- and ozone-induced biochemical changes in
antioxidant enzymes of Arabidopsis thaliana // Plant Physiol. 1996. V. 110. P.125-136.
[19]. Halliwell B. Free radicals, antioxidants and human disease curiosity, cause, or consequence // Lancet.
1994. V.344. P. 721-724. [20]. Fridovich I. Biological effects of the superoxide radical // Arch. Biochem.
1986. V.247. №1. P. 1-11.
[21]. Lushchak V, Gospodaryov. Catalases protect cellular proteins from oxidative modification in
Saccharomyces cerevisiae // Cell Biol Int. 2005. V. 29. Р.187-192.
[22]. Ross R. Atherosclerosis an inflammatory disease // N. Engl. J. Med. 1999. V.340. P. 115-126.
[23]. Jankowski O. J. Oxygen toxicity and biological systems (evolutionary, ecological and medical-biological
aspects) // SPb.: Game. 2000. P. 294.
[24]. Stoker R. Keaney J.F. Role of oxidations in atherosclerosis // Physiol. Rev. 2004. V. 84. P. 1381-1478.
[25]. Trilis J. G., Mescheryakova M. G., Alpatova T. F., Mukhin I. A., Kirillova N. V. The intensity of
oxidative stress in the synovial fluid of patients with osteoarthritis of the knee // Butlerov coomunications.
2012. T. 29. No. 2. P. 109-114.
46 _______ http://butlerov.com/ _______ ©Butlerov Communications. 2015. Vol.43. No.8. P.42-46. (English Preprint)