Page 88 - 79_02

A.
Gómez
et
col.

referred
to
that
of
the
non-­-oxidized
base
(deoxyguanosine,
dG)
by
HPLC-­-EC
as

previously
described
(31).

2.5.
Measurement
of
mitochondrial
complexes
I
to
IV,
AIF,
SIRT1,
PGC1,
TFAM,

Nrf2
and
ERK

The
amounts
of
a)
the
mitochondrial
respiratory
chain
complexes
(I
to
IV),

the
complex
I
regulatory
factor
AIF,
the
mitochondrial
biogenesis
protein

indicators
SIRT1,
PGC1,
and
TFAM,
the
Nrf2-­-ARE
pathway
marker
Nrf2,
and

ERK1/2
pathway
marker
(ERK1/2
and
phospho-­-ERK1/2)
were
estimated
using

western
blot
analyses
as
previously
described
(2).

2.6.
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.

2.7.
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)].

2.8.
Statistics

Values
were
expressed
as
means
±
standard
error
of
the
mean
(SEM).

Comparisons
between
groups
were
analyzed
by
ANOVA
followed
by
DMS
tests
for

paired
groups
and
Student-­-t
test.
The
survival
curve
was
performed
by
Kaplan-­-
Meier
in
Statgraphics,
and
mean
and
maximum
(last
10%
surviving
individuals)

life
span
values
were
analysed
by
the
log
rank
test.
The
minimum
level
of

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

258