Mechanisms of Ageing and Development
98 (1997) 177 – 180

Lipid peroxidation and antioxidant enzymes in
livers and brains of aged rats
S. Dogru-Abbasoglu, S. Tamer-Toptani, B. Ugurnal,
˘
˘
˘
N. Kocak-Toker *, G. Aykac-Toker, M. Uysal
¸
¸
Department of Biochemistry, Istanbul Faculty of Medicine, Uni6ersity of Istanbul, Capa,
¸
34390 Istanbul, Turkey
Received 22 October 1996; accepted 1 May 1997

Abstract
The contribution of free radical damage to aging in the liver and brain is still controversial. There have also been several reports with conﬂicting results on the antioxidant system
during aging. In this study, we investigated endogenous lipid peroxide levels in the liver and
brain tissues of rats aged 6 and 22 months together with ascorbate-induced lipid peroxidation. Also, superoxide dismutase (SOD) and glutathione peroxidase (GPx), the main antioxidant enzymes were assayed. Although ascorbate-induced lipid peroxide levels remained
unchanged in aged animals, hepatic lipid peroxidation was seen to be elevated. Glutathione
(GSH) content was found to be decreased, but SOD and GPx remained unchanged. No
apparent difference in any parameter in brain tissues was observed in the old group. © 1997
Elsevier Science Ireland Ltd.
Keywords: Aging; Glutathione; Glutathione peroxidase; superoxide dismutase; lipid peroxidation

* Corresponding author.
0047-6374/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved.
PII S 0 0 4 7 - 6 3 7 4 ( 9 7 ) 0 0 0 8 2 - 1

178

S. Dogru-Abbasoglu et al. / Mechanisms of Ageing and De6elopment 98 (1997) 177–180
˘
˘

1. Introduction
The free radical theory proposed by Harman, postulates that free radical damage
contributes to the aging phenomenon [1]. However, the results of studies investigating hepatic and cerebral lipid peroxidation in aged animals are still controversial.
Some studies have revealed that there is a signiﬁcant increase in lipid peroxides of
liver and brain homogenates [2,3]. Contrarily some investigators failed to conﬁrm
these results [4,5]. Furthermore, there have been several reports on the antioxidant
system during aging, the results of which are conﬂicting [6,7].
Previously we have observed that lipid peroxidation in homogenates and subcellular fractions in the liver has been increased in aged mice [8]. However, no
signiﬁcant change was observed in lipid peroxide levels in brain homogenates [9]. In
the present study, we aimed to investigate liver and brain lipid peroxidation,
together with ascorbic acid induced lipid peroxidation in aged rats. We also
determined antioxidant enzymes, SOD and GPx in both tissues.

2. Materials and methods
Male Wistar rats aged 6 and 22 months were used in this study. The animals were
starved overnight and killed by decapitation. Livers and brains were rapidly
removed, washed in ice-cold 0.9% NaCl and kept in ice. Tissue homogenates were
prepared with ice-cold 0.15 M KCl (10% w/v). Postmitochondrial fractions were
obtained by centrifugation.
Lipid peroxidation was evaluated as the concentration of thiobarbituric acid
(TBA) by the method of Buege and Aust [10] in liver and brain homogenates.
Results were expressed as nmol malondialdehyde (MDA)/mg protein.The breakdown product 1,1,3,3-tetraethoxypropane was used as standard. Protein concentrations were determined according to the method of Lowry et al. [11].
Ascorbate-induced lipid peroxide levels were also determined in brain and liver
homogenates [12]. The incubation mixture (1 ml) contained 50mM FeSO4, 1 mM
KH2PO4, 0.15M Tris – HCl buffer (pH 7.4), 0.4 mM ascorbic acid and 0.1 ml tissue
homogenate (5 mg protein/ml). After incubation at 37°C in a shaking water bath
for 30 min, MDA formed was estimated according to Buege and Aust [10].
Tissue GSH levels were measured with Ellman’s reagent [13]. SOD activity was
determined in postmitochondrial fractions by the method of Misra and Fridovich
[14]. GPx activity of postmitochondrial fractions was assayed according to Hawkes
and Craig [15]. Results of both enzymatic assays were expressed as U/mg protein.
Statistical analysis was performed by Student’s t-test.

3. Results
Endogenous and ascorbate-induced lipid peroxide and GSH levels and SOD and
GPx activities in liver and brain tissues are shown in Table 1. Although ascorbate-

S. Dogru-Abbasoglu et al. / Mechanisms of Ageing and De6elopment 98 (1997) 177–180
˘
˘

179

induced MDA levels remained unchanged in the old group, hepatic MDA levels
were found to be increased. Hepatic GSH content was found to be decreased in the
old group. However, SOD and GPx activities remained unchanged. On the other
hand, no apparent changes in endogenous and ascorbate-induced lipid peroxide
and GSH levels as well as SOD and GPx activities in the brain were observed in the
old group as compared to the young group.

4. Discussion
It has been suggested that aging involves an increased susceptibility to free
radical reactions [1]. However, in aging tissues of rats such as the liver and brain,
results obtained are contradictory. Unchanged [7,16], decreased [4,5] or high lipid
peroxide levels in the liver and brain [2,8,17] have been reported in old rats
compared to young rats. In this study, liver lipid peroxide content was found to be
increased in aged rats, but no change was observed in brain homogenates, the
results of which were in accordance with our previous studies [8,9]. As for
measuring susceptibility to in vitro lipid peroxidation, decreases of in vitro TBA
values in old rat livers [16] and no changes in brains [16] have been reported. In our
study, no changes in in vitro TBA values were observed in aged rat livers and
brains.
On the other hand, conﬂicting results on the antioxidant system are also
reported. Liver and brain GSH contents have not been found to have changed
[7,18], or have been found to be decreased [2,19]. In this study, liver GSH content
has been seen to be decreased, but brain GSH levels were not found to have
changed.
Table 1
Endogenous and ascorbate-induced lipid peroxide and glutathione levels and superoxide dismutase and
glutathione peroxidase activities in the liver and brain of aged rats
6 months (n =12)

22 months (n =12)

Liver
Malondialdehyde (nmol/mg protein)
Ascorbate-induced lipid peroxide (nmol/mg protein)
Glutathione (nmol/mg protein)
Superoxide dismutase (U/mg protein)
Glutathione peroxidase (mU/mg protein)

0.193 9 0.027
8.53 9 1.08
29.33 9 3.09
10.35 92.44
102.60 9 19.19

0.245 9 0.037**
8.47 9 1.25
23.47 9 2.70*
9.87 9 2.78
97.47 9 15.37

Brain
Malondialdehyde (nmol/mg protein)
Ascorbate-induced lipid peroxide (nmol/mg protein)
Glutathione (nmol/mg protein)
Superoxide dismutase (U/mg protein)
Glutathione peroxidase (mU/mg protein)

0.385 90.075
11.21 91.18
17.56 92.23
3.62 91.20
2.29 9 0.58

Values represent mean 9 S.D.
* PB0.001; ** PB0.01.

0.413 9 0.088
11.77 9 2.08
16.98 9 3.51
4.55 9 0.93
2.51 9 0.77

180

S. Dogru-Abbasoglu et al. / Mechanisms of Ageing and De6elopment 98 (1997) 177–180
˘
˘

SOD and GPx, being fundamental enzymes of antioxidant process, have also
been studied. As for SOD activity, liver and brain SOD activities were unchanged.
There are several reports in the literature of decreased hepatic SOD activity [4,6,20].
On the other hand, Barja de Quiroga et al. [7] have found both liver and brain SOD
activities unchanged. Liver GPx activities were also reported as being decreased [4],
increased [20] or unchanged [6,7]. Brain GPx activities were reported to be
unchanged [4,7]. In our study, liver and brain GPx were also found to be
unchanged. Our results, when considered with the data reported in the literature,
show us that nothing signiﬁcant may be suggested about the changes in antioxidant
enzyme activities and susceptibility to peroxidation in aged rat tissues.

References
[1] D. Harman, Free radical theory of aging: the ‘free radical’ diseases, Age 7 (1984) 111 – 131.
[2] M.Y.H. Farooqui, W.W. Day, D.M. Zamorano, Glutathione and lipid peroxidation in the aging
rat, Comp. Biochem. Physiol. 88B (1987) 177 – 180.
[3] M. Sawada, J.C. Carlson, Changes in superoxide radical and lipid peroxide formation in the brain,
heart and liver during the lifetime of the rat, Mech. Ageing Dev. 41 (1987) 125 – 137.
[4] F. Cand, J. Verdetti, Superoxide dismutase, glutathione peroxidase, catalase, and lipid peroxidation
in the major organs of the aging rats, Free Radic. Biol. Med. 7 (1989) 59 – 63.
[5] H. Zhan, C.P. Sun, C.G. Liu, J.H. Zhou, Age-related change of free radical generation in liver and
sex glands of rats, Mech. Ageing Dev. 62 (1992) 111 – 116.
[6] M.C. Carrillo, S. Kanai, Y. Sato, K. Kitani, Age-related changes in antioxidant enzyme activities
are region and organ, as well as sex, selective in the rat, Mech. Ageing Dev. 65 (1992) 187 – 198.
[7] G. Barja de Quiroga, R. Perez-Campo, M. Lopez-Torres, Anti-oxidant defences and lipid peroxida´
tion in liver and brain of aged rats, Biochem. J. 272 (1990) 247 – 250.
8
[8] M. Uysal, S. Seckin, N. Kocak-Toker, H. Oz, Increased hepatic lipid peroxidation in aged mice,
¸
¸
Mech. Ageing Dev. 48 (1989) 85–89.
8
8
[9] M. Uysal, N. Kocak-Toker, G. Kutalp, G. Ozdemirler, H. Oz, Lipid peroxide levels and Na – K –
¸
ATPase activities in brain homogenates from young and old mice, Turk. J. Med. Biol. Res. 2 (1991)
47–50.
[10] J.A. Buege, J.D. Aust, Microsomal lipid peroxidation, Methods Enzymol. 52 (1978) 302 – 310.
[11] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin
phenol reagent, J. Biol. Chem. 193 (1951) 265 – 275.
[12] T.P.A. Devasagayam, C.K. Pushpendran, J. Eapen, Differences in lipid peroxidation of rat liver
rough and smooth microsomes, Biochem. Biophys. Acta 750 (1983) 91 – 97.
[13] G.L. Ellman, Tissue sulfhydryl groups, Arch. Biochem. Biophys. 82 (1959) 70 – 77.
[14] H.P. Misra, I. Fridovich, Superoxide dismutase: ‘positive’ spectrophotometric assays, Anal.
Biochem. 79 (1977) 553–560.
[15] W.C. Hawkes, K.A. Craig, Automated continuous-ﬂow colorimetric determination of glutathione
peroxidase with dicholoroindophenol, Anal. Biochem. 186 (1990) 46 – 52.
[16] M. Lopez-Torres, R. Perez-Campo, C. Rojas, C. Barja de Quiroga, Sensitivity to in vitro lipid
´
peroxidation in liver and brain of aged rats, Rev. Esp. Fisiol. 48 (1992) 191 – 196.
[17] T. Miyazawa, T. Suzuki, K. Fujimoto, Age-dependent accumulation of phosphatidyl choline
hydroperoxide in the brain and liver of rat, Lipids 28 (1993) 789 – 793.
[18] L.E. Rikans, D.R. Moore, Effect of aging on aqueous-phase antioxidants in tissues of male Fischer
rats, Biochim. Biophys. Acta 966 (1988) 269 – 275.
[19] G.A. Hazelton, C.A. Lang, Glutathione contents of tissues in the aging mouse, Biochem. J. 188
(1980) 25–30.
[20] L.L. Ji, D. Dillon, E. Wu, Alteration of antioxidant enzymes with aging in rat skeletal muscle and
liver, Am. J. Physiol. 258 (1990) R918 – R923.

