Alexia Gómez & col.
618
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)=((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)).
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-‐
