Plasma Malondialdehyde Level and Erythrocytes Catalase Activity

in patients with Type 2 Diabetes Mellitus

Kyae Mhon Htwe1, Mya Thu2, Pyone Pyone Than3
1. Department of Medical Research (Upper Myanmar)
2. University of Nursing, Yangon
3. University of Medicine, Mandalay
Abstract
Lipid peroxidation is the oxidative destruction of lipids by free radicals. The evidence of lipid
peroxidation within biological system is usually determined by malondialdehyde (MDA)
formation. Diabetes mellitus is associated with increased lipid peroxidation and impaired
antioxidant defense mechanisms which exacerbate oxidative stress. Therefore, this study was
aimed to study plasma malondialdehyde level and erythrocytes catalase activity in patients with
type 2 diabetes mellitus and controls. In this study, 30 type 2 diabetes mellitus patients from
diabetic clinic at Mandalay General Hospital and 30 apparently healthy controls were studied.
The subjects were female, 35-50 years of age. The plasma MDA was determined for lipid
peroxidation. The erythrocytes catalase activity was represented as antioxidant enzyme. The
mean plasma MDA level (9.7 4.88 mol/L) in patients with type 2 diabetes mellitus was
significantly higher than that of controls (4.09 2.17 mol/L) (p< 0.001) and mean erythrocytes
catalase activity (2332.84 936.54U/G Hb) in patients was significantly higher than that of
controls (976.59 591.28 U/G Hb) (p< 0.001). Positive correlation between plasma MDA and
erythrocytes catalase in type 2 diabetes mellitus patients (r = 0.62) (p<0.01) was found. These
results suggested that diabetes mellitus is associated with enhanced lipid peroxidation and
increased activities of antioxidant enzymes in erythrocytes to combat the oxidative stress. Hence,
the present study revealed that there is increased oxidative stress in diabetes mellitus which may
be due to increased lipid peroxidation and the body tries to compensate the oxidative stress by
raising the activities of antioxidant enzymes.
Introduction

Diabetes mellitus is not only a simple disorder of carbohydrate metabolism but also
accompanied by various degenerated manifestations such as accelerated aging, cardiovascular
disease and microvascular lesion leading to complications [1]. Generation of free radicals is
accelerated by different mechanisms in diabetes mellitus. Oxidative stress is probably amplified
by a continuous cycle of metabolic stress, tissue damage and cell death due to infection leading
to further radical production [2].
The oxidative destruction of lipids by the free radicals is known as lipid peroxidation.
The membranes that surround the cells and subcellular organelles contain large amount of PUFA
side chains. The peroxidation of PUFAs is initiated by a free radical, such as hydroxyl radical,
which extracts a hydrogen from a polyunsaturated lipid resulting in the formation of a lipid
radical. The free radical chain reaction is propagated by the addition of O2, which forms the lipid
peroxyl radical and lipid peroxide. Degradation of lipid results from rearrangements of the single
electron. Malondialdehyde (MDA), one of the compounds formed in lipid degradation, is soluble
and appears in the blood. The chain reaction can be terminated by antioxidants [3]. Diabetes
mellitus is associated with increased lipid peroxidation. Increased levels of lipid peroxides have
been implicated in the pathogenesis of diabetic complications [4].
MDA is in many instances the most abundant individual aldehyde resulting from lipid
peroxidation. MDA arises largely from peroxidation of PUFAs with more than two double
bonds, such as linolenic, arachidonic and docosa-hexaenoic acids. MDA is readily metabolized
in mammalian tissues. MDA reacts with DNA bases and can introduce mutagenic lesions. MDA
reacts both reversibly and irreversibly with proteins and phospholipids with profound effects. It
is important in diabetes mellitus because the initial modification of collagen by sugar adducts
forms a series of glycation products which then stimulate breakdown of the lipid to MDA and
further cross linking by MDA occurred [5].
Antioxidants inhibiting lipid peroxidation can be classified into preventive antioxidants
that reduce chain initiation (e.g. catalase) and chain-breaking antioxidants that interfere with chain
propagation (e.g. tocopherol, ascorbic acid) [6]. Catalase belongs to oxidoreductase group of
enzymes classified by International Union of Biochemistry. The major active component of the
enzyme, ferriprotoporphyrin (haematin), which occurs one in each subunit, is responsible for
catalyzing 2H2O2 to O2 and 2 H2O. Each subunit usually has one molecule of NADPH bound to it.
The enzyme catalase (EC 1.11.1.6) has a predominant role in controlling the concentration of H 2O2
2

in human erythrocytes [7]. Human erythrocytes with high catalase content provide a general
defense against toxic concentrations of H2O2. H2O2 is involved in physiological processes but its
increased concentration may contribute to the pathogenesis of various diseases including diabetes
mellitus [8]. Therefore, it is important to assess the plasma MDA as parameter for lipid
peroxidation and erythrocytes catalase activity as antioxidant parameter in management of
diabetes mellitus. This study was aimed to investigate the changes in plasma MDA level and
erythrocytes catalase activity in patients with type 2 diabetes mellitus in comparison to normal
controls.
Specific Objectives
1. To determine plasma malondialdehyde level and erythrocytes catalase activity in both patients
with type 2 diabetes mellitus and controls
2. To compare these parameters between two groups
3. To correlate plasma malondialdehyde level and erythrocytes catalase activity in patients with
type 2 diabetes mellitus
Methodology

Study Design
This study was a laboratory based, comparative study.

Study Site
Research was conducted in the Biochemistry Department, University of Medicine, Mandalay.

Study Period
This study was done from June 2007 to December 2007.

Subject Selection
Cases: 30 type 2 diabetes mellitus patients

Inclusion criteria
(1) Patients were between 35-50 years of age
(2) Female patients
(3) Patients diagnosed as type 2 diabetes mellitus (FBS 126 mg /dl) and regularly
attending to diabetic clinic in Mandalay General Hospital (MGH)
(4) Patients who had blood pressure

140/90 mmHg

(5) Non-smokers
3

 Exclusion Criteria
(1) Patients taking regular vitamins supplements such as vitamin E and C
(2) Patients with pregnancy
(3) Patients with other chronic inflammatory diseases
Controls: 30 apparently healthy subjects
Inclusion criteria
(1) Subjects were between 35-50 years of age
(2) Female subjects
(3) No history of diseases and physical examinations revealed nothing abnormal
(5) Subjects who had normal FBS level (80-100mg/dl)
(6) Subjects who had blood pressure

140/90 mmHg

(7) Non-smokers
Exclusion Criteria
(1) Subjects taking regular vitamins supplements such as vitamin E and C
(2) Pregnant women

Operational definition
Non-smoker
Non-smoker means that one who does not smoke at any time.

Sample size calculation

The number of subjects required to provide optimal ability to detect changes in
plasma MDA level and erythrocytes catalase activity were calculated by following
formula for a study using unpairedt test. Two-sided significance level (1-alpha) was
0.05 and power (1-beta, % chance of detecting) was 80.
Standard difference = /
= the assumed equal standard deviation of the observations in each group
= the smallest difference in means that is clinically important
Using Altmans normogram, the line connecting a standard difference and power of 80%
cut the sample size axis (N) approximately and the required sample size in each group was

obtained by N/2 [9]. So, sample size in this study was chosen as 30 subjects for each group
(cases and controls).

Methods
After taking written consent, subjects were interviewed, and history taking and
clinical examinations were done.

Blood sampling
After overnight fasting, about 5 ml of blood was collected into two clean and dry test
tubes from each subject. For FBS determination, 2cc of blood was collected in the test tube
containing sodium fluoride and potassium oxalate as anticoagulant. For plasma MDA and
erythrocytes catalase determinations, 3cc of blood was collected in the test tube containing
double oxalate as anticoagulant. For erythrocytes catalase activity determination, haemolysate
was prepared after removing plasma.
Biochemical methods
Plasma MDA level was determined by using thiobarbituric acid reaction test [10].
Erythrocytes catalase activity was determined by using spectrophotometric method [11].
Hemoglobin concentration was determined by using cyanmethemoglobin method [12]. FBS was
determined by using direct Otoluidine method [13].

Data collection and analysis

Data were recorded according to the proforma. Results were reported as mean
SD. Studentt test (unpaired) was used to observe the significance of difference between above
parameters (MDA and catalase) in type 2 diabetes mellitus patients and controls. The Pearson
correlation was determined.
Results
Thirty type 2 diabetes mellitus patients from diabetic clinic at MGH and thirty apparently
healthy controls were studied. The subjects were females aged between 35-50 years. The plasma
malondialdehyde level and erythrocytes catalase activity in both type 2 diabetes mellitus patients
and controls were determined. The values were expressed as mean SD.
5

Table 1 shows the physical data of type 2 diabetes mellitus patients and controls.
Table 1. The physical data of type 2 diabetes mellitus patients and controls (mean SD)
age in years

FBS (mg/ dl)

systolic blood

diastolic blood

pressure (mmHg) pressure (mmHg)

diabetes mellitus
45.1 2.89
175.64 14.29
131.67 10.91
80.67 6.71
controls
42.23 3.32
75.68 11.94
115.67 9.66
76.55 8.82
Table 2 represents the mean SD of plasma malondialdehyde level and erythrocytes
catalase activity in type 2 diabetes mellitus and controls. Type 2 diabetes mellitus subjects were
found to have significantly higher plasma MDA level and erythrocytes catalase activity than
those of controls (p< 0.001).
Table 2. The plasma MDA level and erythrocytes catalase activity in type 2 diabetes mellitus and
controls
Parameters
plasma
MDA
(mol/L)

level

erythrocytes
catalase
activity (U/G Hb)

diabetes mellitus (n=30)

controls (n=30)

level of significance

9.7 4.88

4.09 2.17

p< 0.001

2332.84 936.54

976.59 591.28

p< 0.001

The results were presented as (mean SD). P < 0.05 was considered as significant.

12
10

9.7

8
6

4.09

4
2
0

Diabetes Mellitus controls

Plas
ma
malo
ndial
dehy
de
(m
ol/L)

Figure 1. The mean plasma MDA in diabetes mellitus and controls

Figure 1 shows the mean plasma MDA levels in patients with type 2 diabetes mellitus
and controls. The mean plasma MDA level in patients with type 2 diabetes mellitus was found to
be significantly higher than that of controls (p< 0.001).
Er
yth
roc
yte
s
cat
ala
se
(U/
G
Hb
)

2500

2332.84

2000

1500
1000

976.59

500
0

Diabetes Mellitus Controls

Figure 2. The mean erythrocytes catalase activity in diabetes mellitus and controls
7

Figure 2 shows the mean erythrocytes catalase activity in patients with type 2 diabetes
mellitus and controls. The mean erythrocytes catalase activity in patients with type 2 diabetes

Plasma MDA level (mol/L)

mellitus was found to be significantly higher than that of controls (p< 0.001).

18
16
14
12
10
8
6

Erythrocytes catalase activity (U/G Hb)

4
2
0

500 1000 15002000 2500 3000 35004000 4500

Figure 3. The correlation between plasma MDA and erythrocytes catalase activity in
patients with type 2 diabetes mellitus
Figure 3 shows the positive correlation between plasma MDA level and erythrocytes
catalase activity in type 2 diabetes mellitus patients (r = 0.62) (r 2= 0.39). The correlation was
significant at p<0.01 level (y = 0.0032x + 2.26).

Discussion
Type 2 diabetes mellitus is one of the major health problems globally that affects over
124 million individuals worldwide. It affects at least 5% of the population in the industrialized
8

world [14]. Excessive production of free radicals observed in type 2 diabetes and its insufficient
removal results in damage to cellular proteins, membrane lipids and nucleic acids. Increased
levels of lipid peroxides have been implicated in the pathogenesis of diabetic complications [4].
Reactive oxygen compounds such as superoxide and hydroxyl radicals have very short half lives.
Therefore, direct measurement of ROS in tissues of diabetic subjects or animals is virtually
impossible. The assessment of MDA has become the most common technique to measure the
degree of oxidative damage in biological systems [15].
In the present study, the mean plasma MDA levels of control subjects and type 2 diabetic
patients were found to be 4.09 2.17mol/L and 9.7 4.88 mol/L. The mean plasma MDA
level of type 2 diabetic patients was found to be significantly higher than controls (p<0.001). In
the study of MDA in type 2 diabetes mellitus with and without complications, excessive
peroxide-mediated damage may appear early on in type 2 diabetes mellitus, before development
of secondary complications [16]. A group of researchers from Turkey found that erythrocytes
and plasma MDA levels of patients with and without retinopathy was significantly higher than
normal controls (p<0.001) [17]. In Myanmar, Chaw Chaw Lynn Sanda found that mean plasma
MDA level was significantly higher in diabetes mellitus (p<0.01) [18]. She also found that there
was correlation between lipid peroxidation and diabetes nephropathy. These studies strongly
suggested that the presence of enhanced oxidative stress in type 2 diabetes mellitus. Therefore,
the present study revealed that there is highly significant association of oxidative stress with type
2 diabetes mellitus probably due to increased lipid peroxidation and it may contribute to diabetic
complications.
The non enzymatic antioxidants e.g., ascorbic acid, tocopherol, -carotene and
enzymatic defenses e.g., catalase, glutathione peroxidase can offer an indication of the
antioxidant status of an individual [19].
The mean erythrocytes catalase activity of control subjects in the present study was
976.59 591.28 U/G Hb. This result falls within the normal reference range of erythrocytes
catalase activity (600-1000 U/G Hb) [20]. The mean erythrocytes catalase activity in patients
was 2332.84 936.54 U/G Hb. Mean erythrocytes catalase activity in patients was found to be
significantly higher than that of controls (p< 0.001). Results of mean erythrocytes catalase
activities in the course of diabetes in various studies were controversial. In the study from
Turkey, an increase in catalase activity was found in patients with diabetes mellitus than controls
9

(p<0.01).

There was positive correlation between catalase activity and FBS level.

They

suggested that uncontrolled glucose metabolism may also be the cause of alterations in
antioxidant enzymes [21]. However, a group of researchers from Poland found that erythrocytes
catalase activities in both patients were significantly lower than control subjects (p< 0.0001).
They suggested that cells adapt to oxidative stress; but under hyperglycemic condition,
progressive glycation of the enzymatic proteins leads to decreases in their activities [22]. These
discrepancies may be partly due to the variability in the diabetes mellitus patients, including age
and sex of diabetes, the severity of the resulting insulin deficiency, and the duration of diabetes.
The antioxidant enzymes (e.g. catalase) protect the cell from oxidative stress but the threshold of
protection can vary dramatically as a function of their activity and balance [23].
Theoretical causes of antioxidant enzyme activity alterations in diabetes may be due to
three mechanisms. First, induction of antioxidant enzyme expression was caused by oxidative
stress. Second, hyperglycemia, enhances non-enzymatic binding of glucose to proteins
(glycation) causes structural and functional changes in the antioxidant enzymes. Thirdly, diabetes
may influence antioxidant enzyme activity through disturbances in micronutrient status. Catalase
contains heme as cofactor. The distribution and function of ions of vital importance may change
in diabetes. Changes in iron metabolism lead to changes at the cofactor level [24].
In the present study, significant positive correlation between plasma MDA and
erythrocyte catalase activity was found (r = 0.62) (p< 0.01). Similar correlation was found in the
studies of other investigators [23, 25] and the present study agreed with these findings. The
antioxidant enzymes (e.g. catalase) protect the cell from oxidative stress but the threshold of
protection can vary dramatically as a function of their activity and balance [23]. These results
strongly supported the hypothesis that in diabetes mellitus, there was increased lipid peroxidation
due to increased free radical production and there was also increased the activities of antioxidant
enzymes in erythrocyte to combat lipid peroxidation. It may be due to the fact that erythrocytes
catalase activity was important in catabolising H2O2. Increasing lipid peroxidation result in
increasing H2O2 formation. So, there is increase in the synthesis and activity of antioxidant
enzyme in the body to prevent oxidative stress.
In conclusion, the present study revealed that diabetes mellitus is associated with
enhanced lipid peroxidation. The increased activity of erythrocytes catalase activity indicated
that the body tries to compensate lipid peroxidation by raising the antioxidant activity.
10

Therefore, plasma malondialdehyde level and erythrocytes catalase activity in type 2 diabetes
mellitus patients could be of importance in the progression of diabetes mellitus and in the
prevention of the development of diabetic complications.
Acknowledgement
I would like to express my grateful thanks to Dr. Daw Khin Than Aye, Associate
Professor/ Consultant, Medical Unit II, MGH for her constructive advice and generous help in
collection of patients. I must also express my profound gratitude to Professor Dr. Daw May
Pyone Kyaw, Head of Biochemistry Department, University of Medicine, Mandalay and
Professor Dr. Daw Khin Win Sein, Head of Biochemistry Department, University of Medicine,
Magway for their patient guidance and suggestions. Finally, I must not fail to mention my
sincere thanks to Dr. Kyaw Zin Thant, Director General, Department of Medical Research
(Upper Myanmar) for his thoughtful help and encouragement.
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