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Age related changes in lipid peroxidation in rat brain and liver
Vol.
78, No.
2, 1977
BIOCHEMICAL
AGE RELATED CHANGES TN LIPID 0.
AND
BIOPHYSICAL
PEROXIDATION LIVER*
RESEARCH
COMMUNICATIONS
TN RAT BRAIN AND
P. SHAlhU
Division of Biochemistry Central Drug Reseeroh Institute, Lucknow - 226001, India. Received
July
12,1977
SUMMARY
Lipid peroxidation in rat wide variations with age* In undergoes wide variations and between ascorbic acid content labile antioxidant factors are There is inverse relationship lipid peroxidation in ljvero
liver, unlike In brain shows liver, ascorbic acid content also there is negative correlation and lipid peroxidation@ Heatpresent In the cytosol fraction. between antioxidant activity and
INTRODUCTION The accumulation evidence Vance
for
--in viva
of lipid
stressed
as a function
lipid
peroxide
peroxidation
by others
investigate
of lipopigments
the of
(2). lipid
in .The
the
present
peroxidation
Is considered formation egeing studies system
(1). process were in rat
to be the The relehas been carried brain
out
to
end liver
age.
EXPERIMENTAL Male albino rats of different age groups drawn from the C.D,R,I, stock colony were fasted overnight (with water --ad libiBrain and liver were taken out, waahed turn) and decapitated. with chilled IFi0 mM KC1 and homogenates (10% W/v) prepared in 150 mh! KC10 Subcellular fractionation was done as described previously (3). 106,000 g supernetant has been designated as the cytosol fraction. For essay of lipid peroxides lo0 ml aliquots of tissue homogenates (or other reaction mixtures) were incubated at 37 + l°C in a metabolic shaker (120 strokes/rain, amplitude I Cm) for-3 hours, the amount of lipid peroxides formed was estimated by the thiobarbituric acid reaction and results have been expressed es malonyldialdehyde (MDA) using an extinction coefficient of lo56x105 at 535 nm (4). Protein estimation was done * Communication No.2294 from the Institute, Lucknow - 226001.
Central
Urug
Research
Vol. 78, No. 2, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table
i
Phospholipid and iron contents brain and liver with Increase (Mean 2 S.D.) Lipid Brain 0
. : :
Pi(mg/iOO gm) : Liver
96.8:1,9
of rat in age ~ -.-_ (mg/iOO gm) : Liver
Iron Brain
131.0+14~0
1.9+_0.2
25.2+6.6
15
141.7+13.1
126e2212.7
1.320.2
5.621.4
25
136,9+17,6
iO2.82
lo620.2
8,3+1.2
30
i46,6+2io4
t04e9zQe 6
1,9+p.l
0.9+1*3
40
162e92904
130,7;ti3.6
2.120.4
8.420.8
60
168 0I+7 .O
149,0~12,6
3.5~1.6
9.e1.4
179.2+15,3
16S.6~14,9
2‘1~0.4
9.9+2,9
240
903
-The data
are mean of eix
experiments.
according to Lowry et al. (5) , asaorbic acid aacording to Roe and Kuether (6) andiron according to Fortune and Mellon (7). Lipids were extracted by the method of Folch and Coworker8 (8) and lipid phosphorus estimated as described earlier (9). Ascorbic acid oxidase (EC ic10.3c3) was prepared from bottle gourd (Lagenaria eiceraria) and assayed by the method of total antioxidant Lovett-Janison and Nelson (IO) 0 For assaying activity due to antioxidant enzymes like euperoxide dismutase (II), glutathione peroxidase- (12) and semidehydroascorbate dehydrogenase (13) amount of lipid peroxides formed by unheated and heated (for 5 minutes in boiling water bath) tissue homogenates was estimated and the results have been expressed ae percent increase in lipid peroxide formation on inactivating the labile antioxidant enzymes, RES ULTS
AM)
D ISCDSS ION
Rates of shown in Fig.
lipid
peroxidation
I. Phospholipid
liver
are shown in Table
brain
of 0 day old
age of the animal
animals
and ascorbic and iron Amount
I.
is rather
and reaches
of
acid
are
brain
and
content
in rat
lipid
peroxides
in the
increases
with
low,
it
a maximum in the
470
content
the
20 days old rate,
Vol.
78, No.
2, 1977
0
BIOCHEMICAL
60 AGRDAYS)
Fig.
120
thereafter
RESEARCH
COMMUNICATIONS
240 AGCCDAYS)
it
content
in
does
brain
age of
the
marked
alterations.
the
BIOPHYSICAL
- Lipid peroxidation and ascorbic acid content of rat brain and liver (Mean + SIT),) M Lipid peroxidation; M Ascorbic acid
i
tion
AND
in
not is
animal.
the
any
quite
low
Iron
content
On the
liver
suckling
show
shows
rats
marked
at
birth in
other
wide
(0-y 9O days),
a maximum range
months,
it
declines
brain
does
is
weaning
and reaches
forma-
quite
in
low
(21 days)
the
the
undergo
peroxide
It
during
with
not
lipid
ions*
after
ases and maintains thereafter
and increases
hand,
Yariat
Phospholipid
variation0
it
incre-
age of
one to two
level
of newborn
the
animals o On treating
tissue
homogenates
S3.6$
and 0S,i$
inhibition
liver
and brain
respectively,
brain
and liver
is apparently
is heat-labile ship
between
activity
is
increases level
and is lipid evident
of peroxide
two to three
liver0 fold
ascorbic formation
The antioxidant due to dismutase
shown in Fig*
peroxide for
with
formation
(Fig*
e
471
30 days
oxidase
was noticed
in
activity
of rat
reaction
which
2 and an inverse
Ant ioxidant within
acid
relation-
1) and antioxidant activity and remains
in brain at
this
Vol. 78, No. 2, 1977
BIOCHEMICAL
activity 2 - Antioxidant (Mean + S,Do) e-----e
Figo
Studies activity
on subcellular
indicate
free
fraction
rat,s
is less
that
(Table
On the
rats9 animals
rvations
correlation
animals
lipid
analysis
developing
role
for
ascorbic
have been takon
tion
between
rat8
rats.
mitochondrial
there
antioxidant
for
of lipid
acid
acid
present brain
experimentatfonc p8roxidationo
472
as old on addi-
The Same obsefrom
fractions. in Fig.
is negative
action
in rat
as well
suapewions
presented
and ascorbic
However,
(14).
formation
peroxidation
has been Attributed
brain
that
lipid
1 month old
show that
a non-enzymic
such a role
peroxide
from young
of the data
peroxidation
A similar
from 6 month old
when added to the cytosol
coefficient
implies
liv8ro
microsomes
from
particulate
from 6 months or 1 month old
in undergoing
fraction
Statistical
It
hand,
antioxidant
the
fraction lipid
microsomes
have been made with
young and old
between
with
the
with
The cytosol in promoting
do not differ
of
iS associated
2).
other
of cytosol
in rat brain and liver Brain; o----o Liver.
localization
it
efficient
when reconstituted
tion
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
correlation
content
in liver.
of ascorbic to ascorbic
Studies
I for
acid acid
in in
do not suggest
in the age groups There ascorbic
is
no correla-
acid,
phospho-
Vol. 78, No. 2, 1977
BIOCHEMICAL
AND BIOPHYSICAL
Table Lipid
peroxidation
RESEARCH COMMUNICATIONS
2
in young
and old rats.
-BP
Reaction
n mole Malonyl dlaldehyde/ml reaction mixture/3hr
mixture
Cytosol
(I)
+ 3dicrosomes (4.)
Cytosol
(6)
+ Micr*somes
(I)
5.6
Cytosol
(I)
+ hlierosomes
(6)
14.6
Cytosol
(6)
+ Mlcrosomes
(6)
7.5
14.3
The reaction mixture (Oe’f5 ml) contained cytoeol (106,OOr) g supernatant, 3*22 mg protein) and microsonal suspension in 150 !nU KC1 (690 ug protein), In parentheses the age of the animal in month 4 18 given0
lipid
and iron
content
brain*
In consonance
Stewart
(15)
brain
acid,
vitamin
lipid
peroxide
Ascorbic
acid
peroxidation,
of young
formation oxidase
workers
(14,
Low level
and liver
the
presented
9 imply
(16,
17).
of
lipid
inhibition
a dual
upon its
of
1) show an These observa-
in liver* role
has been ascribed
depending
mediator
above (Fig.
acid
and of ascorbic
amounts
natural
in
peroxident
to ascorbic
concentration
as acid
by
in the
159 19). of antioxidants
special
propensity
Frinna
and Barber
peroxides
about
ascorbic
that
brain
activity
of Allison
had higher
in rat brings
of
as antioxidant
tissues
rats
C has been shown to be the
action
earlier
antioxidant
the observations
al t,hough paradoxical
well the
with
The results
antioxidant tions,
and enzymatic
in older
in brain
to accumulate (21)
lipopigments
have also
animals.
may contribute
observed
In liver,
473
with
lower
output
to
ageing
values
of lipid
its (20),
of lipid peroxi-
Vol. 78, No. 2, 1977
de8 appears tic
to be subtly
antioxidants
peroxidation to
its
young tion
lity is
{Fig.
role
I,
lipofuscin lipid
obscure0
It
the liver
an inbuilt
The wide
The reason (I)
may also
in lipid
may have relevance
and disposition
(22)
the reported
in the
old animals
inspiteof
antioxidant
activity
be surmised
mechanism against
and erdyma-
for
and high
animals
acid
variations
in liver
metabolism
peroxides
of old
by ascorbic
levels
pigments
of
of
2).
in drug
and old animals, of
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
regulated
and antioxidant
special
low levels still
BIOCHEMICAL
to
that
undergo
the adveree
lower lipid effects
in
accumulathe is
susceptlbiperoxidation of ageing
process a ACKNOWLEDGWENTS The author is grateful to Dr. C.R, Krishna Murti for guidance and inspiration, to Dro O.P. Shukla for helpful discussions and critical perusal of the manuscript and to the Council of Scientific and Industr’ial Research, New Delhi, India for the award of a Research Fellowship. REFERENCES 1, 2-0
3. 4r 5. 6.
Tappel, &L. (1979) Fed* Proc.(F.ASEB Meeting} p. 239, Minoru, O,, Manfred, S, and Mario, B, (1969) Blood, 34, 712-716 0 Schneider, W.C, and Hogeboom, G,Ho (1950) 3. Biol. Chem. 183, 123-128. Bernheim, Fc and Hochstein, P. (1967) Arch, Utley, H.G,, Biochem. Biophye,, 118, 29-32. Lowry O.H., Rosebzgh, N,J., Farr, A.L. and Randall, R,J. (1951) J. Riol, Chem. 193, 265-275, Roe, J,Ho and Kuether,TA* (1943) Jo Biol, Chem. 147, 399-407 W.B, and Nbllon, M.Go (1957) cited in Methods in Fortune, Enzymology, Vole 3p p. 1017 (Edited by S,P, Colowick and N. 0. Kaplan) o G.Ho (1957) J, Folch, J., Lees, M. and SloaneSt.anley, Biol* Chemo 226, 497-509. Rouser, G,, motes, A.N, and Fleischer, So (1966) lipids, A., 85-86. Covett-Janison, P.Le and Nelson, J.M. (1940) Jo Amero Chem. sot, 62, 1409-1412. I. (1975) Ann* Rev. Biochemo 44, 147-159. Fridovich, Christopheraen, B.0. (1969) Biochimo Biophys, Acta. 176, 463-470. Green, R,C. and O’Brien, P.J, (1973) Blochim, Biophys. dcta, 293, 334-342. Ad1ard,8,P,F. (1974) Biochemc Soce Trans. 2, 281-284, l
70 8. 9. IO. il. 12c 134 140
474
Vol. 78, No. 2, 1977
15. 16. 17* 16, 190 20. 21. 22.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Allison, J,H. and Stewart, MoA, (1973) Jo Neurochem. z, 1705-1786. Sharma, O.P, and Krishna Murti, COB, (1976) Jo hmchem. 27, 299-301, Barber, Aode (1966) Lipids, & 146-1st. Hunter, F.E. Jr*, Soott, A,, Hoffstein, P.E., Guerra, F., Weinstein J., Schneider, Ao, Schutz, B,, Fink, J. and Ford, L. (1964) J, Biolo Chem. 239, 604-613. Sharma, O.P, (1977) Indian Jc Bioche& Riophye, (In prese), (1969) in Pigments In Pathology Porta, E.AA, and Hartfort edited by W. Wolman, New York, Academic Pre8S, pp 191, Grinna, L.S, and Barber, &,A. (1973) Blochem* Biophys, Reso Commun* 55, 773-779, Schacter, B,A., Marver, HA, and Meyer, U,A, (1973) Drug Metaso Disposition, & 286-290.
475
78, No.
2, 1977
BIOCHEMICAL
AGE RELATED CHANGES TN LIPID 0.
AND
BIOPHYSICAL
PEROXIDATION LIVER*
RESEARCH
COMMUNICATIONS
TN RAT BRAIN AND
P. SHAlhU
Division of Biochemistry Central Drug Reseeroh Institute, Lucknow - 226001, India. Received
July
12,1977
SUMMARY
Lipid peroxidation in rat wide variations with age* In undergoes wide variations and between ascorbic acid content labile antioxidant factors are There is inverse relationship lipid peroxidation in ljvero
liver, unlike In brain shows liver, ascorbic acid content also there is negative correlation and lipid peroxidation@ Heatpresent In the cytosol fraction. between antioxidant activity and
INTRODUCTION The accumulation evidence Vance
for
--in viva
of lipid
stressed
as a function
lipid
peroxide
peroxidation
by others
investigate
of lipopigments
the of
(2). lipid
in .The
the
present
peroxidation
Is considered formation egeing studies system
(1). process were in rat
to be the The relehas been carried brain
out
to
end liver
age.
EXPERIMENTAL Male albino rats of different age groups drawn from the C.D,R,I, stock colony were fasted overnight (with water --ad libiBrain and liver were taken out, waahed turn) and decapitated. with chilled IFi0 mM KC1 and homogenates (10% W/v) prepared in 150 mh! KC10 Subcellular fractionation was done as described previously (3). 106,000 g supernetant has been designated as the cytosol fraction. For essay of lipid peroxides lo0 ml aliquots of tissue homogenates (or other reaction mixtures) were incubated at 37 + l°C in a metabolic shaker (120 strokes/rain, amplitude I Cm) for-3 hours, the amount of lipid peroxides formed was estimated by the thiobarbituric acid reaction and results have been expressed es malonyldialdehyde (MDA) using an extinction coefficient of lo56x105 at 535 nm (4). Protein estimation was done * Communication No.2294 from the Institute, Lucknow - 226001.
Central
Urug
Research
Vol. 78, No. 2, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table
i
Phospholipid and iron contents brain and liver with Increase (Mean 2 S.D.) Lipid Brain 0
. : :
Pi(mg/iOO gm) : Liver
96.8:1,9
of rat in age ~ -.-_ (mg/iOO gm) : Liver
Iron Brain
131.0+14~0
1.9+_0.2
25.2+6.6
15
141.7+13.1
126e2212.7
1.320.2
5.621.4
25
136,9+17,6
iO2.82
lo620.2
8,3+1.2
30
i46,6+2io4
t04e9zQe 6
1,9+p.l
0.9+1*3
40
162e92904
130,7;ti3.6
2.120.4
8.420.8
60
168 0I+7 .O
149,0~12,6
3.5~1.6
9.e1.4
179.2+15,3
16S.6~14,9
2‘1~0.4
9.9+2,9
240
903
-The data
are mean of eix
experiments.
according to Lowry et al. (5) , asaorbic acid aacording to Roe and Kuether (6) andiron according to Fortune and Mellon (7). Lipids were extracted by the method of Folch and Coworker8 (8) and lipid phosphorus estimated as described earlier (9). Ascorbic acid oxidase (EC ic10.3c3) was prepared from bottle gourd (Lagenaria eiceraria) and assayed by the method of total antioxidant Lovett-Janison and Nelson (IO) 0 For assaying activity due to antioxidant enzymes like euperoxide dismutase (II), glutathione peroxidase- (12) and semidehydroascorbate dehydrogenase (13) amount of lipid peroxides formed by unheated and heated (for 5 minutes in boiling water bath) tissue homogenates was estimated and the results have been expressed ae percent increase in lipid peroxide formation on inactivating the labile antioxidant enzymes, RES ULTS
AM)
D ISCDSS ION
Rates of shown in Fig.
lipid
peroxidation
I. Phospholipid
liver
are shown in Table
brain
of 0 day old
age of the animal
animals
and ascorbic and iron Amount
I.
is rather
and reaches
of
acid
are
brain
and
content
in rat
lipid
peroxides
in the
increases
with
low,
it
a maximum in the
470
content
the
20 days old rate,
Vol.
78, No.
2, 1977
0
BIOCHEMICAL
60 AGRDAYS)
Fig.
120
thereafter
RESEARCH
COMMUNICATIONS
240 AGCCDAYS)
it
content
in
does
brain
age of
the
marked
alterations.
the
BIOPHYSICAL
- Lipid peroxidation and ascorbic acid content of rat brain and liver (Mean + SIT),) M Lipid peroxidation; M Ascorbic acid
i
tion
AND
in
not is
animal.
the
any
quite
low
Iron
content
On the
liver
suckling
show
shows
rats
marked
at
birth in
other
wide
(0-y 9O days),
a maximum range
months,
it
declines
brain
does
is
weaning
and reaches
forma-
quite
in
low
(21 days)
the
the
undergo
peroxide
It
during
with
not
lipid
ions*
after
ases and maintains thereafter
and increases
hand,
Yariat
Phospholipid
variation0
it
incre-
age of
one to two
level
of newborn
the
animals o On treating
tissue
homogenates
S3.6$
and 0S,i$
inhibition
liver
and brain
respectively,
brain
and liver
is apparently
is heat-labile ship
between
activity
is
increases level
and is lipid evident
of peroxide
two to three
liver0 fold
ascorbic formation
The antioxidant due to dismutase
shown in Fig*
peroxide for
with
formation
(Fig*
e
471
30 days
oxidase
was noticed
in
activity
of rat
reaction
which
2 and an inverse
Ant ioxidant within
acid
relation-
1) and antioxidant activity and remains
in brain at
this
Vol. 78, No. 2, 1977
BIOCHEMICAL
activity 2 - Antioxidant (Mean + S,Do) e-----e
Figo
Studies activity
on subcellular
indicate
free
fraction
rat,s
is less
that
(Table
On the
rats9 animals
rvations
correlation
animals
lipid
analysis
developing
role
for
ascorbic
have been takon
tion
between
rat8
rats.
mitochondrial
there
antioxidant
for
of lipid
acid
acid
present brain
experimentatfonc p8roxidationo
472
as old on addi-
The Same obsefrom
fractions. in Fig.
is negative
action
in rat
as well
suapewions
presented
and ascorbic
However,
(14).
formation
peroxidation
has been Attributed
brain
that
lipid
1 month old
show that
a non-enzymic
such a role
peroxide
from young
of the data
peroxidation
A similar
from 6 month old
when added to the cytosol
coefficient
implies
liv8ro
microsomes
from
particulate
from 6 months or 1 month old
in undergoing
fraction
Statistical
It
hand,
antioxidant
the
fraction lipid
microsomes
have been made with
young and old
between
with
the
with
The cytosol in promoting
do not differ
of
iS associated
2).
other
of cytosol
in rat brain and liver Brain; o----o Liver.
localization
it
efficient
when reconstituted
tion
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
correlation
content
in liver.
of ascorbic to ascorbic
Studies
I for
acid acid
in in
do not suggest
in the age groups There ascorbic
is
no correla-
acid,
phospho-
Vol. 78, No. 2, 1977
BIOCHEMICAL
AND BIOPHYSICAL
Table Lipid
peroxidation
RESEARCH COMMUNICATIONS
2
in young
and old rats.
-BP
Reaction
n mole Malonyl dlaldehyde/ml reaction mixture/3hr
mixture
Cytosol
(I)
+ 3dicrosomes (4.)
Cytosol
(6)
+ Micr*somes
(I)
5.6
Cytosol
(I)
+ hlierosomes
(6)
14.6
Cytosol
(6)
+ Mlcrosomes
(6)
7.5
14.3
The reaction mixture (Oe’f5 ml) contained cytoeol (106,OOr) g supernatant, 3*22 mg protein) and microsonal suspension in 150 !nU KC1 (690 ug protein), In parentheses the age of the animal in month 4 18 given0
lipid
and iron
content
brain*
In consonance
Stewart
(15)
brain
acid,
vitamin
lipid
peroxide
Ascorbic
acid
peroxidation,
of young
formation oxidase
workers
(14,
Low level
and liver
the
presented
9 imply
(16,
17).
of
lipid
inhibition
a dual
upon its
of
1) show an These observa-
in liver* role
has been ascribed
depending
mediator
above (Fig.
acid
and of ascorbic
amounts
natural
in
peroxident
to ascorbic
concentration
as acid
by
in the
159 19). of antioxidants
special
propensity
Frinna
and Barber
peroxides
about
ascorbic
that
brain
activity
of Allison
had higher
in rat brings
of
as antioxidant
tissues
rats
C has been shown to be the
action
earlier
antioxidant
the observations
al t,hough paradoxical
well the
with
The results
antioxidant tions,
and enzymatic
in older
in brain
to accumulate (21)
lipopigments
have also
animals.
may contribute
observed
In liver,
473
with
lower
output
to
ageing
values
of lipid
its (20),
of lipid peroxi-
Vol. 78, No. 2, 1977
de8 appears tic
to be subtly
antioxidants
peroxidation to
its
young tion
lity is
{Fig.
role
I,
lipofuscin lipid
obscure0
It
the liver
an inbuilt
The wide
The reason (I)
may also
in lipid
may have relevance
and disposition
(22)
the reported
in the
old animals
inspiteof
antioxidant
activity
be surmised
mechanism against
and erdyma-
for
and high
animals
acid
variations
in liver
metabolism
peroxides
of old
by ascorbic
levels
pigments
of
of
2).
in drug
and old animals, of
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
regulated
and antioxidant
special
low levels still
BIOCHEMICAL
to
that
undergo
the adveree
lower lipid effects
in
accumulathe is
susceptlbiperoxidation of ageing
process a ACKNOWLEDGWENTS The author is grateful to Dr. C.R, Krishna Murti for guidance and inspiration, to Dro O.P. Shukla for helpful discussions and critical perusal of the manuscript and to the Council of Scientific and Industr’ial Research, New Delhi, India for the award of a Research Fellowship. REFERENCES 1, 2-0
3. 4r 5. 6.
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