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Experimental Gerontology 36 (2001) 209±220

209

www.elsevier.nl/locate/expgero

Alterations of antioxidant enzymes and oxidative
stress markers in aging q
È
M. Kasapoglu, T. Ozben*
Medical Faculty, Department of Biochemistry, Akdeniz University, 07058 Antalya, Turkey
Received 16 June 2000; received in revised form 30 August 2000; accepted 30 August 2000

Abstract
In accordance with the present state of scienti®c knowledge, the excessive production of free
radicals in the organism, and the imbalance between the concentrations of these and the antioxidant
defenses may be related to processes such as aging and several diseases. The aging process has been
described by various theories. In particular, the free radical theory of aging has received widespread
attention which proposes that deleterious actions of free radicals are responsible for the functional
deterioration associated with aging. Although, the relationship between lipid peroxidation and aging
have been investigated extensively, the studies have produced con¯icting results. To investigate the
correlation between the oxidative stress and aging, we have determined the levels of lipid peroxidation expressed as thiobarbituric acid reactive substances (TBARS; MDA) and conjugated dien;
oxidative protein damage as indicated by carbonyl content and activities of antioxidant enzymes
including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in a
sample of 100 healthy men and women ranging in age from 20 to 70 years. In addition, vitamin
E, C levels, reduced glutathione and sulphydryl content were determined. The oxidation end product
of nitric oxide (nitrate) was also studied to investigate any role of nitrogen radicals in aging. Our data
show that there is an age related increase in lipid peroxidation expressed as MDA and oxidative
protein damage as indicated by carbonyl content. Aging is not linked to a decline in antioxidant
enzymes except GPx. Our data suggests that the level of oxidative stress increase cannot entirely be
attributed to a decrease in the activities of antioxidant defense system and probably various factors
may contribute to this process. q 2001 Elsevier Science Inc. All rights reserved.
Keywords: Aging; Antioxidants; Oxidative stress markers

q
Presented in part at the SFRR-Europe Meeting in Dresden, 2±5 July 1999.
* Corresponding author. Tel.: 190-242-227-59-96; fax: 190-242-227-49-58.
È
E-mail address: ozben@akdeniz.edu.tr (T. Ozben).

0531-5565/01/$ - see front matter q 2001 Elsevier Science Inc. All rights reserved.
PII: S 0531-556 5(00)00198-4

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1. Introduction
Aging is an inevitable biological process that affects most living organisms. Despite the
enormous consequences associated with the aging process, until recently, relatively little
systematic effort has been expended on the scienti®c understanding of this important life
process. Society, however, urged by an ever increasing older population, is challenging
scientists from many disciplines to explore one of the nature's most complex phenomenabiological aging. For the past two decades, research directed toward the basic understanding of biological aging mechanisms and possible aging interventions have given us new
insights into the molecular bases and the biological events that contribute to age-related
deterioration (Yu, 1996).
Aging is ªa genetic physiological process associated with morphological and functional
changes in cellular and extracellular components aggravated by injury throughout life and
resulting in a progressive imbalance of the control regulatory systems of the organism,
including the hormonal, autocrine, neuroendocrine, and immune homeostatic mechanismsº (Yu, 1996). Aging contributes to the susceptibility to disease and one dies from a
distinct pathological event, not the aging process (Ramesh, 1987; Timiras, 1994; Forbes,
1997; Harman, 1991; Harman, 1992a,b). In general, aging is characteristically described
as a time-dependent functional decline, leading to the cell's incapacity to withstand
external and internal challenges. According to this description, aging is the consequence
of two independent biological processes: the loss of functionality, and the loss of resistance or adaptability to stress. The causal factors that underlie the time-dependent, deleterious processes of aging have not yet been well de®ned, and no single adequate
molecular explanation for aging is currently available. The consensus among researchers
broadly views the concept of biological aging as an organism's failure to maintain homeostasis (Gutteridge, 1992). The contributions of the aging process to aging changes are
small early in life but rapidly increase with age due to the exponential nature of the process
(Ramesh, 1987; Timiras, 1994; Forbes, 1997; Harman, 1991; Harman, 1992a,b).
According to a recent account from Medvedev, more than 300 theories on aging exist
(Medvedev, 1990). No one theory is generally accepted. The free radical theory of aging was
proposed in November 1954 by Harman which proposes that aging results from imperfect
protection against tissue damage brought about by free radicals. This aging theory is meritorious for having provided modern gerontologists with both a conceptual base and the
experimental opportunities necessary to explain the biological basis of senescence and
disease (Yu, 1996; Strehler, 1980; Comfort, 1979; Yu and Yang, 1996; Freeman, 1984;
Boje and Arora, 1992; Masters, 1994; Moncada et al., 1992). However, a major obstacle
to accept the theory has been the poor record of antioxidants in prolonging the life span of
animals. Many other variables, such as genetic factors, temperature, activity and nutrition can
affect life span, making it a highly complex multi-factorial process.
The participation of free radical reactions in the pathogenesis of many diseases is now
generally accepted. Whether or not these reactions are responsible for aging is still being
debated. Although, the relationship between lipid peroxidation and aging has been investigated extensively, the studies have produced con¯icting results (Bermejo and Hidalgo,
1997; Casado et al., 1996; Artur et al., 1992; Rikans and Hornbrook, 1997; Beckman and
Ames, 1998; Ashok and Ali, 1999).

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211

The aim of this work is to study the relationship between the lipid peroxidation
expressed as thiobarbituric acid reactive substances (TBARS; MDA) and conjugated
dien; oxidative protein damage as indicated by carbonyl content and activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT) and glutathione
peroxidase (GPx) in healthy human subjects during the aging process. In addition, vitamin
E, C levels, reduced glutathione (GSH) and sulphydryl content were determined. The
oxidation end product of nitric oxide (nitrate) was also studied to investigate any role
of nitrogen radicals in aging.
2. Materials and methods
Fasting blood samples of 100 healthy individuals in ®ve age groups (ten male and ten
female, in each group), age range between 20±29; 30±39; 40±49; 50±59 and 60±69 were
obtained from a group of volunteers. Only those who proved to be in a normal state of
health and free from any signs of chronicle disease by a careful clinical examination,
biochemical and hematological analysis were included in the study. They were nonsmokers and not consuming alcohol regularly. Most of the subjects were not receiving
any drug on a long-term basis. The intake of analgesics, or anti-in¯amatuars (very few
subjects) was cut a few weeks before blood sampling. Serum was obtained by centrifugation at 1500g for 15 min of blood samples taken without anti-coagulant.
Thiobarbituric acid reactive substances in serum were determined by the ¯uorometric
method of Wasowicz et al. (1993) and the results were expressed as nmol/l MDA. Spectrophotometric detection of lipid conjugated dienes was determined by the method of
Recknagel and Glende (1984). Serum sulphydryl concentrations were determined by
the method of Koster et al. (1986). Carbonyl content in oxidatively modi®ed proteins
was determined by the method of Levine et al. (1990). The absorbance of the sample was
measured at 360 nm and the results were given as mmol carbonyl/l by using e max
22,000 M 21 cm 21. Serum vitamin E concentration was measured as described by Desai
(1984). Concentrations were calculated using a standard curve of known concentrations of
d-a-tocopherol acetate. Vitamin C (McCormick, 1986) concentrations were measured in
serum by the method of McCormick. Ascorbic acid is oxidized by Cu 12 to form dehydroascorbic acid which reacts with acidic 2,4-dinitrophenylhydrazine to form a red bishydrazone which is measured at 520 nm. Serum nitrate was measured by an enzymatic
one-step assay with nitrate reductase. Following reduction to nitrite by nitrate reductase,
serum nitrate concentration is measured by the decrease in absorbance at 340 nm as a
result of oxidation of NADPH (Ohta et al., 1986; Bories and Bories, 1995). Erythrocyte
reduced glutathione concentration was assayed by the method of Fairbanks and Klee
(1986). The results were expressed as mg/gHb. In the hemolysates obtained as mentioned
below, the enzymatic activities of SOD (Misra and Fridovich, 1972); CAT (Aebi, 1983)
and GPx (Paglia and Valentine, 1967) were assayed. The results were expressed as U/gHb
for SOD and GPx; and as k/gHb for CAT. Erythrocyte Cu, Zn-SOD activity was assayed
by the spectrophotometric indirect inhibition technique of Misra and Fridovich based on
the ability of SOD to inhibit the autooxidation of adrenalin to adrenochrome at alkaline pH
(Misra and Fridovich, 1972). CAT activity was measured in erythrocytes by the method of

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Fig. 1. Variations in the mean values of the serum MDA levels as a function of age for men and women. Vertical
lines correspond to SEM. *p , 0.05 Signi®cantly different from age group 50±59.

Aebi using hydrogen peroxide as a substrate. The method is based on the decomposition of
hydrogen peroxide which is indicated by the decrease in absorbance at 240 nm (Aebi,
1983). GPx activity was measured in erythrocytes by the coupled method of Paglia and
Valentine using t-butyl hydroperoxide as a substrate (Paglia and Valentine, 1967).
2.1. Preparation of the hemolysates
The whole blood samples were collected in vacuum tubes containing 1.7 mg/ml
K3EDTA as anticoagulant. After centrifugation at 1500g for 15 min the plasma and
buffy coat were removed. Then, red blood cells were washed twice in an ice-cold isotonic
sodium chloride solution (1:10, v/v) and the packed cells obtained were resuspended in the
washing solution to give a 50% suspension. Hemolysis of the washed cell suspension was
achieved by mixing 1 vol. with 9 vol. of distilled water. The hemolysate obtained was
divided into two aliquats. One aliquat was used to determine the hemoglobin concentration
using the cyanomethemoglobin method (Tentori and Salvati, 1981). The second aliquat
was used for the spectrophotometric determination of the enzymatic activities of SOD,
CAT and GPx.
2.2. Statistical analysis
The statistical analysis was performed using multiple regression analysis. Pearson's
correlation coef®cient (r) was computed to test the association between the different
variables and age. Mean values were calculated per decade. Variations in the mean values
of measured parameters were represented in the ®gures as a function of age for gender.
Vertical lines correspond to the SEMs in the various age groups of population. For each
subgroup, mean values were compared with that of other aged groups. The 0.05 level was
selected as the point of minimal statistical signi®cance (Tukey studentized range method).

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213

Fig. 2. Variations in the mean values of the serum conjugated diene levels as a function of age for men and
women. Vertical lines correspond to SEM.

3. Results
In our study, we found no evidence that gender, weight, blood pressure in¯uenced
signi®cantly the parameters measured in the same and different age groups. Since there
was no gender difference, the combined values of men and women in different age groups
were compared with each other.
Our data show that there is an age related increase in the concentration of lipid peroxides, with men showing higher or about equal values until about 60 years, after which aged
women show slightly higher values, although not signi®cantly different (Fig. 1). The mean
MDA values were signi®cantly higher in the age group 50±59 as compared with that of
30±39 age group. Serum conjugated diene levels did not change with age (Fig. 2). On the
other hand, there was a generalized signi®cant decrease in serum sulphydryl levels with
age (Fig. 3). There was a signi®cant difference between the mean sulphydryl level at

Fig. 3. Variations in the mean values of the serum sulphydryls levels as a function of age for men and women.
Vertical lines correspond to SEM. *p , 0.05 Signi®cantly different from age groups 30±39, 40±49, 50±59 and
60±69.

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M. Kasapoglu, T. Ozben / Experimental Gerontology 36 (2001) 209±220

Fig. 4. Variations in the mean values of the serum carbonyl contents as a function of age for men and women.
Vertical lines correspond to SEM. *p , 0.05 Signi®cantly different from age group 60±69.

20±29 as compared to those at 30±39, 40±49, 50±59 and 60±69 age groups. Protein
carbonyl content, an indicator of oxidative protein damage, increased in an age-related
pattern with signi®cantly higher levels in 60±69 age group compared to 20±29 and 30±39
age groups (Fig. 4).
The enzymatic activities in red blood cells also show an age-dependent behavior. SOD
activity after age group 30±39 reached signi®cantly higher values at age groups for 50±59
and 60±69(Fig. 5). Similarly, there was a constant increase in CAT levels and signi®cantly
high levels were found in the age group of 60±69 as compared to that of 20±29 age group
(Fig. 6). GPx was the only enzyme which decreased with aging (Fig. 7). The lowest
signi®cant level was obtained at the age group of 60±69 as compared with the 20±29
age group. We found no signi®cant difference in the activities of these three enzymes with
gender.
Our data show that aging is not associated with a change in the mean values of GSH

Fig. 5. Variations in the mean values of the erythrocyte SOD activities as a function of age for men and women.
Vertical lines correspond to SEM. *p , 0.05 Signi®cantly different from age groups 50±59 and 60±69.

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M. Kasapoglu, T. Ozben / Experimental Gerontology 36 (2001) 209±220

215

Fig. 6. Variations in the mean values of the erythrocyte CAT activities as a function of age for men and women.
Vertical lines correspond to SEM. *p , 0.05 Signi®cantly different from age group 60±69.

(Fig. 8). Vitamin C and E levels did not change with age and there were also no gender
difference (Figs. 9 and 10).
The nitrate level showed an age-related increase and mean values of groups 5 and 4
were signi®cantly higher than those of groups 1, 2 and 3 (Fig. 11).
4. Discussion
Our data support the interpretation that the level of oxidative stress increases during the
aging process. Is it related due to a decline in antioxidant defenses or an increase in the rate
of prooxidant generation? A perusal of literature indicates a chaotic lack of agreement.
Activities of the same enzymes in the same tissue of the same species are reported to go up
or down or remain unchanged during aging (Sohal and Orr, 1992).
In our subjects, aging was linked to an increase in antioxidant enzymes, SOD and CAT,

Fig. 7. Variations in the mean values of the erythrocyte GPx activities as a function of age for men and women.
Vertical lines correspond to SEM. *p , 0.05 Signi®cantly different from age group 60±69.

216

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M. Kasapoglu, T. Ozben / Experimental Gerontology 36 (2001) 209±220

Fig. 8. Variations in the mean values of the erythrocyte GSH levels as a function of age for men and women.
Vertical lines correspond to SEM.

except GPx. The only signi®cant decrease in GPx levels was observed only between age
groups of 60±69 and 20±29. In other age groups, mean GPx values were not statistically
different. This was in accordance with our ®nding of similar reduced glutathione levels in
all age groups.
Aerobic cells contain various amounts of the three main antioxidant enzymes: SOD,
CAT, and GPx. These three enzymes are necessary for cell survival since inhibition of
their activity leads to the arrest of cell mitosis and to cell death. Amongst them, GPx was
shown to be more ef®cient than CAT and much more than SOD. With age, the level of
antioxidant enzymes does not change in several experimental models, so that it is not
possible to explain the aging process by a lack of protection due to a decrease in the
activity of the antioxidant enzymes (Remacle et al., 1992).
The fact that there is no correlation between the CAT and GPx activities with the
maximum life span of various species and that at least in some aging models, there is
no decreased activity, strongly indicates that aging is not associated with any shortage in

Fig. 9. Variations in the mean values of the serum vitamin C levels as a function of age for men and women.
Vertical lines correspond to SEM.

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M. Kasapoglu, T. Ozben / Experimental Gerontology 36 (2001) 209±220

217

Fig. 10. Variations in the mean values of the serum vitamin E levels as a function of age for men and women.
Vertical lines correspond to SEM.

antioxidant enzyme protection at least in these models. What is the reason for the higher
susceptibility of old tissues and cells to free radicals and their higher rate of peroxidation
when cell homogenates are left for spontaneous oxidation.
One possible explanation could be that the enzymes are modi®ed in old tissues and that
their ef®ciency is lowered and they are modi®ed with age (Remacle et al., 1992). Another
explanation may be: If in vivo oxygen radical generation is depressed in old animals, the
decreases of the antioxidant defenses should be interpreted as a physiological compensatory down regulation instead of a deleterious change leading to additional oxidative
damage. Finally, most of the reports concentrate on a single or a few antioxidants. This
can complicate the interpretation of the results since it is known that cellular antioxidants
are under homeostatic control. A decrease in a particular antioxidant can be compensated
by an increase in a different one not included in the study. This is why comprehensive

Fig. 11. Variations in the mean values of the serum nitrate levels as a function of age for men and women. Vertical
lines correspond to SEM. *p , 0.05 Signi®cantly different from age group 50±59 and 60±69. **p , 0.05
Signi®cantly different from age group 60±69.

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studies of the whole antioxidant system during aging are needed (Barja de Quiroga et al.,
1992).
Our data showed that lipid peroxidation increased with aging when expressed as
TBARS, but not as conjugated dien. There was not a generalized increase of TBARS
with age. Statistically signi®cant increases were only seen when comparing selected age
groups, for example, MDA was higher in the 30±39 age group with respect to the 50±59
age group, but not with respect to the other age groups. In literature, several results
regarding the increases or decreases of the levels of thiobarbituric acid with aging have
been reported (Barja de Quiroga et al., 1992). MDA and conjugated dienes are only two
easily detectable products of lipid peroxidation. Poly unsaturated fatty acids (PUFAs)
suffer oxidation into a plethora of different products and at some point it will be important
to get a full spectrum of these products (and their bioactivity) for young and old tissue in
order to decide whether lipid peroxidation is an important contributor to age-related
disorder. MDA test is very sensitive but poor in speci®city. Certainly the most direct
approach for the assessment of lipid peroxidation is the quanti®cation of the primary
(hydroperoxides) products, but in practice, it is very dif®cult because of their labile,
¯eeting nature. Consequently, detection of lipid peroxidation has relied largely on indirect
methods, that is, analyses of secondary or end products derived from hydroperoxides such
as malondialdehyde (MDA).
In our study, protein carbonyl content was found to increase in an age-related pattern
indicating an increase in oxidative protein damage with aging. The increase we observed
in serum carbonyl levels was probably the most impressive ®nding in terms of supporting
the hypothesis.
We observed a generalized signi®cant decrease in serum sulphydryl levels with age. An
important fact is, however, that the measurement of, e.g. total serum thiols may not be
representative for what happens with individual biomolecules. For example, if a critical
thiol of an important protein suffers oxidation (and this protein looses activity), such
modi®cation may not be detected in ªtotal thiol measurementsº unless this particular
protein is highly expressed. In animal models it has been shown that, for example, certain
proteins suffer oxidation of .1 mol cys/mol protein (Viner et al., 1999).
Signi®cantly higher levels of serum nitrate were detected for older age groups which
may indicate higher levels of NO and peroxynitrate, with the latter being a powerful
oxidant.
On the basis of currently available evidence, it is concluded that the free radical
hypothesis has neither been proven nor disproved. There is no key evidence at
present that establishes a direct causal link between oxidative stress and the rate
of aging. All the purported supportive evidence is either correlative or inconclusive
(Lenaz et al., 1998; Knight, 1998; Perez-Campo et al., 1998; Harman, 1998; Sastre
et al., 2000). Similarly, there is no compelling evidence, at this time, to reject the
hypothesis either. Some of the earlier assumptions such as that antioxidant intake
increases life span, or antioxidant defenses decline with age, or antioxidant defenses
are positively correlated with life spans of different species, or that longer life spans
are associated with lower autoxidizability are not clearly supportable (Yu et al.,
1998). Clearly, further studies, some of which speci®cally focus on disproving
this hypothesis, are needed to con®rm its veracity (Sohal, 1993).

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219

Further research is needed to ®nd out if lowering serum peroxide levels of aging
subjects by diet supplementation with antioxidants will decrease that risk. Today, the
aging process remains an enigma, and scienti®c inquiries into the processes and causes
of aging are still in their infancy.
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