Original Paper
Received: May 2, 2003
Ophthalmologica 2004;218:202206
DOI: 10.1159/000076845
Changes in Antioxidant Enzyme Activity
and Malondialdehyde Level in Patients
with Age-Related Macular Degeneration
zlem Yldrm a Nurcan Aras Ates b Llfer Tamer c Necati Muslu c
Bahadr Ercan c Ugur Atik c Arzu Kank d
Departments of a Ophthalmology, b Medical Biology and Genetics, c Biochemistry, and d Biostatistics,
Faculty of Medicine, Mersin University, Mersin, Turkey
Key Words
Age-related macular degeneration W Catalase W
Malondialdehyde W Aging and oxidative stress
Abstract
Age-related macular degeneration is the leading cause
of legal blindness in the developed world, and yet its
pathogenesis remains poorly understood. Oxidative
stress may play a major role in the etiology and patho-
genesis of age-related disorders such as age-related
macular degeneration. Catalase is an antioxidant en-
zyme which plays an important role in the detoxification
of free oxygen radicals. Malondialdehyde is a marker
that shows free radical damage. We have measured the
erythrocyte activity of catalase and the serum level of
malondialdehyde in 30 patients with age-related macular
degeneration and 60 healthy subjects. Patients with age-
related macular degeneration showed significantly low-
er catalase activity compared to healthy subjects (p =
0.002). Plasma malondialdehyde level of the patient
group was significantly higher than that of the controls
(p = 0.038).
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Accepted after revision: October 9, 2003
Introduction
Age-related macular degeneration (AMD) is the lead-
ing cause of visual impairment in the elderly and the most
common cause of blindness in the developed world [1]. Its
prevalence is likely to rise as a consequence of increasing
longevity [2]. As such, it represents a major public health
problem. Its etiopathogenesis cannot be enlightened, al-
though clinical signs and natural course have been known
for a long time.
Epidemiologic studies have suggested that ocular, sys-
temic factors [3, 4] and genetic factors [5] are risk factors
for AMD. However, aging is the only definite risk factor
for AMD.
Free oxygen radicals have been proposed as important
causative agents of aging. The theory that aging increases
because of free radical damage has been suggested by
Harman [6]. The main source of free radicals is molecular
oxygen and these species can cause oxidative damage in
the cell. Aging can thus be viewed as a process of irrevers-
ible injuries associated with accumulation of these oxida-
tive changes [7].
The human retina is a unique component of the ner-
vous system in that throughout life it is continuously
exposed to optical radiation between 400 and 1,400 nm
zlem Yldrm, MD
Mersin niversitesi Tp Fakltesi Hastanesi
Zeytinlibahe Cad.
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[8]. The retina is an ideal environment for the generation
of reactive oxygen species (ROS) for several reasons. First
oxygen consumption by the retina is much greater than by
any other tissue [9]. Second, the retina is subject to high
levels of cumulative irradiation. Third, photoreceptor
outer segment membranes are rich in polyunsaturated fat-
ty acids, which are readily oxidized and which can initiate
a cytotoxic chain reaction [10]. Fourth, the neurosensory
retina and the retinal pigment epithelium (RPE) contain
an abundance of photosensitizers [1113]. Finally, the
process of phagocytosis by the RPE is itself an oxidative
stress and results in the generation of ROS [14].
The biological effects of ROS are controlled in vivo by
a wide spectrum of enzymatic (superoxide dismutase, cat-
alase and glutathione peroxidase) and nonenzymatic (vi-
tamin E, C, carotenoids, metallothionein) defense mecha-
nisms [15]. According to the free radical theory of aging,
one might expect the activity of antioxidant enzymes to
be altered [16]. There are some regulatory mechanisms in
senescent tissues to provide an efficient antioxidant de-
fense against free radicals, which may be generated at a
higher rate during the aging process [17].
Several authors have suggested that the free radical
hypothesis of aging applies to AMD, in particular because
the central area of the neurosensory retina and the un-
derlying RPE may be susceptible to the cumulative toxic
effects of continued photic damage [8, 1820].
In this study, erythrocyte activity of catalase (CAT),
which is among the antioxidant enzymes, and plasma lev-
el of malondialdehyde (MDA), which forms as a result of
lipid peroxidation and is accepted as a marker of free radi-
cal damage, were measured in patients with AMD and
compared with a control group. By this way, it was inves-
tigated whether changes in serum accompany antioxi-
dant-oxidant imbalance which has been proved to occur
in the tissue.
Material and Methods
This study was performed with 30 AMD patients whose ages
ranged from 47 to 82 (12 male, 18 female) attending Mersin Univer-
sity Faculty of Medicine Department of Ophthalmology. Sixty
healthy subjects (30 men, 30 women; age range 4573 years), who
visited our hospital for an annual checkup, were used as a control
group. All subjects were submitted to clinical examination to detect
the signs or symptoms of chronic disease such as arterial hyperten-
sion, diabetes mellitus, chronic anemia, thyroid function disorders,
liver and renal dysfunction, or inflammatory arthritis. Individuals
with chronic disease and/or under drug treatment were excluded
from the study. All subjects were nonsmokers and none was consum-
ing alcohol.
Changes in Catalase and MDA in Patients
with Age-Related Macular Degeneration
After obtaining a detailed ophthalmic and medical history,
complete ophthalmological examination including slitlamp biomi-
croscopy of the anterior segment, applanation tonometry, fundus-
copic examination and fundus fluorescein angiography were per-
formed in all subjects.
The type of AMD was the exudative form, defined on the basis of
the history of illness and ophthalmoscopic and fluorescein angio-
graphic findings including classic or occult choroidal neovascular
membranes, lesions in the RPE such as serous or hemorrhagic RPE
detachments, and fibrovascular disciform degeneration. Familial
cases of AMD and such hereditary diseases as late-onset Best disease
and Sorsby fundus dystrophy were excluded from the study. Thus, a
total of 30 patients with exudative AMD in either one eye or both
eyes were recruited.
Control individuals had no senescent ocular disorders, such as
visually significant cataract. During the examination, the Lens
Opacities Classification System II, which uses photographic stan-
dards for grading cataract type and severity and was described by
Chylack et al. [21], was used to grade lens status with slitlamp exami-
nation. Both lenses of control subjects were graded as having no
nuclear, posterior subcapsular, cortical opacities or having grade I or
II opacities. Also there was no evidence of AMD by fundus examina-
tion. Pathological changes in the RPE, fibrovascular degeneration at
the maculae, and hereditary macular dystrophies were excluded.
Thus, a total of 60 individuals were recruited as controls.
After all subjects gave their informed consent, venous blood sam-
ples (10 ml), taken in ethylenediaminetetraacetic acid as an anticoag-
ulant, were obtained from patients and controls.
Determination of Catalase. Catalase activity was measured ac-
cording to the method described by Beutler [22].
Principle. Catalase catalyzes the breakdown of H2O2 to H2O and
O2. The rate of decomposition of H2O2 by catalase is measured spec-
trophotometrically at 230 nm, since H2O2 absorbs light at this wave-
length. Ethanol is added to stabilize the hemolysate by breaking
down complex II of catalase and H2O2.
Determination of MDA. MDA level as an index of lipid peroxida-
tion was determined by thiobarbituric acid reaction according to
Yagi [23]. The principle of the method depends on measurement of
the pink color produced by interaction of barbituric acid with MDA
elaborated as a result of lipid peroxidation. The color reaction
1,1,3,3-tetraethoxypropane was used as the primary standard [23].
Statistical Method
Analysis of covariance (ANCOVA) taking age as the continuous
covariate and gender as the categorical covariate was evaluated for
difference between AMD and control groups about MDA and CAT,
so the effects of confounders such as age and gender were eliminated.
MDA and CAT values are given as adjusted mean B standard error.
Statistical test was performed by statistical program for social science
(SPSS) version 10 (version 10.0) statistical software program for win-
dows; p value ! 0.05 was considered as statistically significant.
Results
General characteristics of patient and control groups
are summarized in table 1. The mean age of the patient
group was 72.6 B 1.40 and 40% of the patient group was
Ophthalmologica 2004;218:202206 203
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Table 1. General characteristics of patients and control group
Patient group Control group
(n = 30) (n = 60)
Age, years 72.6B1.40 57.4B1.08
Gender, m/f 12/18 30/30
male; 50% of the control group was male with a mean age
of 57.4 B 1.08.
Erythrocyte CAT activity and plasma MDA level are
shown in table 2. CAT activity was found to be 11,819.8
B 1,323.3 U/l in AMD patients and 16,061.3 B 1,126.6
U/l in control cases. The statistically significant relation-
ship was found between age and CAT activity. CAT activ-
ity changed significantly with age (p = 0.027), whereas
there was not a significant difference between genders
from the point of view of CAT activity (p = 0.559). The
effect of age on CAT activity was eliminated during statis-
tical analysis since this effect was found statistically sig-
nificant. CAT activity of AMD patients was significantly
lower than that of the control group (p = 0.002).
A statistically significant relationship was found be-
tween gender and MDA level, but there was no significant
relation between age and MDA level. In other words, it
was determined that gender affects plasma MDA level
(p = 0.031) but variation of age does not affect MDA. So
any effect of gender on MDA level was eliminated. Plas-
ma MDA mean of AMD patients (4.41 B 0.84 nmol/ml)
was significantly higher than that of the controls (3.12 B
0.48 nmol/ml, p = 0.038).
Discussion
The free radical theory of aging proposes that aging
and age-related disorders are the result of cumulative
damage arising from reactions involving ROS [24]. Dur-
ing aging, the balance between the generation of ROS and
ROS clearance can be disturbed resulting in oxidative
damage to macromolecules such as membrane phospho-
lipids [25]. Within the eye, these damaging reactions have
been proposed to be involved in the pathogenesis of AMD
[26]. The precise role of the damaging effects of ROS in
the development and progression of the disorder remains
to be explained.
A hematogenous basis for the development of AMD,
which involves oxidative damage, has been proposed by
204 Ophthalmologica 2004;218:202206
Table 2. Erythrocyte CAT enzyme activity and plasma MDA level
of patient and control groups
Patient group Control group
(n = 30) (n = 60)
CAT activity, U/la 11,819.8B1,323.3 16,061.3B1,126.6
MDA level, nmol/mlb 4.41B0.84 3.12B0.48
a CAT activity of the patient group was significantly lower than
that of the control group (p = 0.002).
b MDA level of the patient group was significantly higher than that
of the control group (p = 0.038).
Gottsch et al. [27], who suggested that development of
AMD is caused by chronic light exposure with direct cho-
riocapillaris damage. Further evidence for involvement of
blood factors is suggested by the finding that a high plas-
ma antioxidant index has a protective effect in AMD [28].
In addition, protection against the development of severe
AMD has been attributed to increased dietary consump-
tion of foods high in carotenoids, which are known to pro-
tect against singlet oxygen-mediated oxidative damage
[29]. The exact role of blood antioxidant substance in dis-
ease development and progression remains to be fully elu-
cidated.
Studies of systemic antioxidant enzyme activities in
AMD patients have led to conflicting results [3034].
These differences may depend on geographical location,
methodology and inadequate attention to confounding
factors such as gender, alcohol consumption, smoking and
various disease.
Previous reports suggest that blood levels of antioxi-
dant enzymes may be lower in individuals with AMD
than in controls. Low glutathione peroxidase [30, 31], glu-
tathione reductase [30], and superoxide dismutase [31]
activities in red blood cells of AMD patients compared
with age-matched controls have been identified. The find-
ing of lower levels of superoxide dismutase and gluta-
thione peroxidase in small series of patients compared
with controls led one group to propose that red blood cells
can be used as a biological index for studying progress of
disease in AMD [31].
De La Paz et al. [32] investigated whether an associa-
tion between AMD severity and particular major antioxi-
dant enzyme levels in red blood cells exists. They found
no evidence for an association between the severity of
aging maculopathy and antioxidant enzyme activity.
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Kank
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 The population-based and cross-sectional study which
investigates the relationship between systemic antioxi-
dant enzyme activities and AMD with the broadest scope
is the POLA study [33]. The POLA study found that high-
er plasma levels of glutathione peroxidase were signifi-
cantly associated with a 9-fold increase in the prevalence
of late AMD, but were unassociated with early AMD.
This finding suggests that glutathione peroxidase is one of
the strongest indicators of AMD ever identified. But in
the POLA study no association could be detected between
systemic superoxide dismutase activity and AMD.
Among the previous studies which investigate the rela-
tionship between AMD and systemic antioxidant enzyme
levels, only De La Paz et al. [32] investigated CAT activi-
ty. In our study, it was determined that systemic catalase
activity changes with age (p = 0.027). Systemic catalase
activity in AMD patients was statistically significantly
lower than in the control group (p = 0.002). It has been
postulated, therefore, that increased oxidative stress asso-
ciated with AMD results in upregulation of erythrocyte
catalase activity. Our results contradict the results of
De La Paz et al. [32] when we consider all AMD cases
included in our study as late AMD cases. Besides, it was
determined that gender affects plasma MDA level (p =
0.031) and that the MDA level of the patient group was
significantly higher than that of the control group (p =
0.038). In this preliminary study, the result of increased
lipid peroxidation in serum samples from AMD patients
is consistent with those of previous studies [3034] in
which age-related decreases in the capacity of antioxidant
systems have been found to be more pronounced in
patients with AMD compared with controls. Significantly
higher MDA level was detected in the plasma of patients
with AMD compared with their controls in the study by
Totan et al. [34], which was performed on patients with
exudative AMD. High polyunsaturated fatty acid content
of photoreceptor membranes particularly exposes the reti-
na to increased risk of lipid peroxidation by unopposed
action of free radicals [35]. It has been suggested that the
retina is very susceptible to lipid peroxidation, and that
this susceptibility also increases with aging in the macular
region [26].
Finally, the decrease in CAT activity and also the high
level of plasma MDA demonstrate the importance of oxi-
dative stress in AMD pathogenesis. Our hypothesis is that
oxidative stress may lead to the induction of antioxidant
enzymes. The results of this preliminary study suggest
that possible alterations of CAT activity and plasma
MDA level are associated with AMD. More data are
needed at the biochemical and epidemiologic levels for a
better understanding of these findings.

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