Page 102 - 79_04

Alexia
Gómez
&
col.

indicators
SIRT3
and
SIRT5
and
MnSOD
were
estimated
using
western
blot

analyses
as
previously
described
(36).

Oxidation-­-derived
protein
damage
markers

GSA,
AASA,
CML,
CEL
and
MDAL
were
determined
as
trifluoroacetic
acid

methyl
esters
(TFAME)
derivatives
in
acid
hydrolyzed
delipidated
and
reduced

mitochondrial
protein
samples
by
GC/MS
(32)
using
a
HP6890
Series
II
gas

chromatograph
(Agilent,
Barcelona,
Spain)
with
a
MSD5973A
Series
detector
and
a

7683
Series
automatic
injector,
a
HP-­-5MS
column
(30-­-m
x
0.25-­-mm
x
0.25-­-µm),

and
the
described
temperature
program
(32).
The
amounts
of
product
were

expressed
as
µmoles
of
GSA,
AASA,
CML,
CEL
or
MDAL
per
mol
of
lysine.

Fatty
acid
analyses
and
global
fatty
acid
unsaturation
indexes

Fatty
acids
from
mitochondrial
lipids
were
analyzed
as
methyl
esters

derivatives
by
gas
chromatography
(GC)
as
previously
described
(33).
The

following
fatty
acyl
indices
were
also
calculated:
saturated
fatty
acids
(SFA);

unsaturated
fatty
acids
(UFA);
monounsaturated
fatty
acids
(MUFA);

polyunsaturated
fatty
acids
(PUFA)
from
n-­-3
and
n-­-6
series
(PUFAn-­-3
and
PUFAn-­-
6);
and
average
chain
length
(ACL)=((S%Total14
x
14)
+
(S%
Total16×16)
+

(S%Total18×18)
+
(S%Total20×20)
+
(S%
Total22×22)
+
(S%
Total24×24))/100.
The

density
of
double
bonds
in
the
membrane
was
calculated
by
the
Double
Bond

Index,
DBI
=
((1×Smol%
monoenoic)
+
(2×Smol%
dienoic)
+
(3×Smol%
trienoic)
+

(4×Smol%
tetraenoic)
+
(5×Smol%
pentaenoic)
+
(6×Smol%
hexaenoic)).
Finally,

the
membrane
susceptibility
to
peroxidation
was
calculated
by
the
Peroxidizability

Index,
PI=
((0.025×Smol%
monoenoic)
+
(1×Smol%
dienoic)
+
(2×Smol%
trienoic)

+
(4×Smol%
tetraenoic)
+
(6×Smol%
pentaenoic)
+
(8×Smol%
hexaenoic)).

Statistics

Data
were
analyzed
by
Student-­-t
tests.
The
minimum
level
of
statistical

significance
was
set
at
P<
0.05
in
all
the
analyses.

3.
RESULTS

The
mean
body
weight
of
the
animals
did
not
show
significant
differences

between
the
two
experimental
groups
at
the
beginning
of
the
experiment
(172

±2.45
g
in
the
control
and
175
±2.67
g
in
the
atenolol
group).
No
significant

differences
in
body
weight
were
observed
after
two
weeks
of
treatment
with

atenolol
either
(299
±4.09
g
in
the
control
and
301
±4.75
g
in
the
atenolol
group).

No
significant
differences
in
heart
weight
and
food
or
water
intake
were
found

between
the
atenolol
and
the
control
groups
(data
not
shown).

The
rate
of
oxygen
consumption
from
heart
mitochondria
was
measured
in

the
absence
(state
4)
and
in
the
presence
(state
3)
of
500µM
ADP,
with
complex
I-­-

618