A. Gómez et col.
258
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)=[(Σ%Total
14
x 14) + (Σ% Total
16
×16) +
(Σ%Total
18
×18) + (Σ%Total
20
×20) + (Σ% Total
22
×22) + (Σ% Total
24
×24)]/100. The
density of double bonds in the membrane was calculated by the Double Bond
Index, DBI = [(1×Σmol% monoenoic) + (2×Σmol% dienoic) + (3×Σmol% trienoic) +
(4×Σmol% tetraenoic) + (5×Σmol% pentaenoic) + (6×Σmol% hexaenoic)]. Finally,
the membrane susceptibility to peroxidation was calculated by the Peroxidizability
Index, PI= [(0.025×Σmol% monoenoic) + (1×Σmol% dienoic) + (2×Σmol% trienoic)
+ (4×Σmol% tetraenoic) + (6×Σmol% pentaenoic) + (8×Σmol% 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.
