HIGH-RESOLUTION RESPIROMETRY OF PERMEABILIZED SKELETAL-MUSCLE FIBERS IN THE DIAGNOSIS OF NEUROMUSCULAR DISORDERS

High resolution respirometry in combination with the skinned fiber tec hnique offers the possibility to study mitochondrial function routinel y in small amounts of human muscle. During a period of 2 years, we inv estigated mitochondrial function in skeletal muscle tissue of 13 patie nts (average age = 5.8 years). In all of them, an open muscle biopsy w as performed for diagnosis of their neuromuscular disorder. Mitochondr ial oxidation rates were measured with a highly sensitive respirometer . Multiple substrate-inhibitor titration was applied for investigation of mitochondrial function. About 50 mg fibers were sufficient to obta in maximal respiratory rates for seven different substrates (pyruvate/ malate, glutamate/malate, octanoylcarnitine/malate, palmitoylcarnitine /malate, succinate, durochinol and ascorbate/TMPD). Decreased respirat ion rates with reference to the wet weight of the permeabilized fiber could immediately be detected during the course of measurements. In 4 patients with mitochondrial encephalomyopathy (MEM) the respiration pa ttern indicated a specific mitochondrial enzyme defect, which was conf irmed in every patient by measurements of the individual enzymes (one patient with PDHC deficiency, one with complex I deficiency and two pa tients with combined complex I and IV deficiency). In the 6 patients w ith spinal muscular atrophy (SMA) oxidation rates were found to be dec reased to 23 +/- 5% of controls. The normalized respiration pattern wa s comparable to that of the controls indicating a decreased content of mitochondria in SMA muscle with normal functional properties. Also in the 3 patients with Duchenne muscular dystrophy (DMD) decreased oxida tion rates (42 +/- 5%) were detected. In addition a low RCI (1.2) indi cated a loose coupling of oxidative phosphorylation in the mitochondri a of these patients. It is concluded that investigation of mitochondri al function in saponin skinned muscle fibers using high resolution res pirometry in combination with multiple substrate titration offers a va luable tool for evaluation of mitochondrial alterations in muscle biop sies of children suffering from neuromuscular disorders.