FUNCTIONAL-CHARACTERIZATION OF MITOCHONDRIAL OXIDATIVE-PHOSPHORYLATION IN SAPONIN-SKINNED HUMAN MUSCLE-FIBERS

The conditions of treatment of human skeletal muscle fibers from M. va stus lateralis with saponin were optimized to achieve complete permeab ilization of cell membrane at intact mitochondrial oxidative phosphory lation. After 30 min of incubation with saponin all lactate dehydrogen ase, 50% of creatine kinase, 30% of adenylate kinase and less then 20% of citrate synthase was released into the permeabilization medium. Th ese skinned fibers behave similar to isolated mitochondria from human skeletal muscle: (i) the respiration with mitochondrial substrates can be stimulated by ADP, (ii) inhibited by carboxyatractyloside and (iii ) it is possible to detect fluorescence changes of mitochondrial NAD(P )H on additions of substrates, uncoupler and cyanide. From a compariso n of rates of respiration per cytochrome aa3 content of isolated human skeletal muscle mitochondria and saponin-skinned muscle fibers it was possible to calculate that almost 85% of mitochondria in those fibers are accessible for the investigation of oxidative phosphorylation. As shown by the investigation of biopsy samples of two patients with und efined myopathies these fibers are a suitable object for the replaceme nt of isolated mitochondria in the diagnosis of mitochondrial myopathi es and encephalomyopathies.