Calcium content and respiratory control index of isolated skeletal muscle mitochondria: effects of different isolation media.
Calcium (Ca) content measured with graphite furnace atomic absorption spectrometry and polarographical measurement of respiratory control index (RCI) were performed on isolated skeletal muscle mitochondria. Four different isolation media were used in order to develop a method for isolating physiologically intact mitochondria in small samples with an endogenous Ca content as close as possible to in vivo conditions. With ruthenium red in the isolation medium, the mean Ca content decreased fourfold to 19.3 +/- 6.8 nmol/mg protein and RCI increased about 25% to 6.9 +/- 1.1 compared with a standard medium. With EGTA or EGTA plus ruthenium red, the mean Ca content decreased further to 5.8 +/- 1.0 and 5.1 +/- 0.9 nmol/mg protein and the mean RCI values increased to 8. 4 +/- 1.2 and 8.8 +/- 1.3, respectively. Isolated mitochondria from human muscle samples using EGTA and ruthenium red in the isolation medium had mean Ca values of 11.0 +/- 0.9 nmol/mg protein and mean RCI values of 12.2 +/- 1.6. The data indicate that the Ca concentration in the skeletal muscle mitochondria is inversely proportional to RCI, and the mitochondrial functional property is significantly improved upon lowering the intramitochondrial Ca accumulation by the incorporation of a chelating agent (EGTA) and a potent Ca antagonist, ruthenium red, in the isolation media.
Madsen
K
K
Department of Physical Education, Odense University, Campusvej 55, Odense M, DK-5230, Denmark.
Ertbjerg
P
P
Pedersen
P K
PK
D000328:Adult; D000818:Animals; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D008297:Male; D008931:Mitochondria, Muscle / Q000378:metabolism*; D010101:Oxygen Consumption; D051381:Rats; D017207:Rats, Sprague-Dawley
D002118:Calcium