Influence of the phospholipid n-6:n-3 polyunsaturated fatty acid ratio on the oxidative phosphorylation of isolated cardiac mitochondria (part 5)
The free calcium concentration in the three buffers was assessed by fluorimetry at 37°C using the fluorescent probe fura-2 (pentapotassium salt). These measurements were realized in the conditions of respiration assessments (with2+palmitoylcarnitine-malate and ADP-Mg ), except that the mitochondria were not added. The excitation and emission wavelengths to quantify the calcium-fura-2 complex were 335 and 505 nm, respectively. They were 363 and 512 nm for the free form of fura-2. The free calcium concentrations, evaluated from the Ca2+-fura-2 complex to total fura-2 ratio, were 0.5, 0.34 and 0 in buffers 1,2 and 3, respectively.
High energy phosphate production: This reaction took place in a 5 mL incubation chamber under the same conditions as the oxygen consumption measurements. The same media, differing in their free calcium concentration, were used. The phosphorylation reaction was initiated by the addition of ADP-Mg2+ (final concentration 333 |j.M) and was maintained for 10 mins at 37°C with 0.33 mg mitochondrial proteins/mL. To ensure enough oxygen supply, the medium was continuously supplied with air. Aliquots were withdrawn at different times following the addition of ADP-Mg2+ (1, 2, 3, 5 and 10mins). They were immediately acidified with perchloric acid (final concentration 0.6 M) and neutralized with KOH and centrifuged (1200 g for 5 mins). The amounts in high energy phosphates (CP, ATP, ADP and AMP) were determined in the supernatants by a one-step high performance liquid chromatography analysis according to Harmsen et al. The energy produced (EP) in the medium was calculated according to the formula: EP = CP + ATP + 1/2 ADP + 1/3 AMP and was expressed in moles equivalent ATP. CP,ATP, ADP and AMP were the amounts ( moles) in high energy phosphate in the medium. The metabolic efficiency (nmoles equivalent ATP/ng atom O ) was the ratio of rate of energy production to rate of state IV oxygen consumption. Best quality drugs cheap: find and discover best deals online.