Influence of the phospholipid n-6:n-3 polyunsaturated fatty acid ratio on the oxidative phosphorylation of isolated cardiac mitochondria (part 3)
ANIMALS AND METHODS
Animals and diets: Weanling male Wistar rats were housed in individual cages in temperature-controlled animal quarters and randomly assigned to two groups. The animals were fed ad libitum for eight weeks a semipurified diet composed of casein (180 g/kg), starch (390 g/kg), sucrose (260 g/kg), salt mixture (50 g/kg), agar (20 g/kg), vitamin mixture and either sunflower seed oil (100 g/kg) (SSO group) or a mixture (1:1 w/w) of fish oil (EPAX 3000TG, Pronova, Bergen, Norway) and sunflower seed oil (100 g/kg) (FO group). The fatty acid composition of the dietary oils is presented in Table 1.
Mitochondria extraction: Heart mitochondria were extracted from ventricles according to the method of Palmer et al with the following modifications. The rats were killed by cervical dislocation and, after a rapid thoracotomy, the heart was removed and immersed in a small volume of cold bufferA(KCl 100 mM, 3-[N-morpholino]propane-sulphonic acid [MOPS] 50 mM, EDTA 2 mM, pH 7.4, bovine serum albumin 0.2%).The extra ventricular tissues were discarded and the myocardium was finely minced with scissors. After suspension in buffer A (2.7 mL/g), the minced ventricles were homogenized with a polytron (2.4 s, rheostat = 3) and a potter-Elvehjem (350 rpm). The homogenate was centrifuged (500 g, 10 mins) and the supernatant 1, containing mechanically extracted mitochondria, was stored at 0 C. The pellet was resuspended in buffer A (2.7 mL/g). Aprotease (nagarse, 5 mg/g) was added and the suspension was homogenized with a potter-Elvehjem (350 rpm). The homogenate was diluted (one:three) with buffer A within 30 s following nagarse addition, and immediately centrifuged (4200 g for 7 mins). The pellet was resuspended in buffer A (5.4 mL/g) and cellular debris separated out by centrifugation (500 g for 10 mins) and discarded. The supernatant containing enzymatically extracted mitochondria was added to supernatant 1. The resulting supernatant was washed twice in buffer B (KCl 100 mM, MOPS 50 mM, EDTA 0.5 mM, pH 7.4, bovine serum-albumin 0.2%) and centrifuged (4000 g for 10 mins). Before the last centrifugation (4000 g for 10 mins), the pellet was washed in buffer B devoid of serum albumin. The mitochondria were then suspended in the last buffer at the approximate concentration of 20 mg/mL. The protein content was determined by the method of Lowry et al. You will love this opportunity to shop with best pharmacy on the internet and pay less every time you visit: to discover exactly how much less you could be spending while still getting your treatment exactly the way you need it.
TABLE 1 Fatty acid composition of the dietary oils
|Fatty acid||Sunflower seed oil||Fish oil|