Acetyl-CoA-driven respiration in frozen muscle contributes to the diagnosis of mitochondrial disease

Background Freezing human biopsies is common in clinical practice for storage. However, this technique disrupts mitochondrial membranes, hampering further analyses of respiratory function. To contribute to laboratorial diagnosis of mitochondrial diseases, this study sought to develop a respirometry approach using O2k (Oroboros Ins.) to measure the whole electron transport chain (ETC) activity in homogenates of frozen skeletal muscle biopsies. Patients and Methods We enrolled 16 patients submitted to muscle biopsy in the process of routine diagnostic investigation: four with mitochondrial disease and severe mitochondrial dysfunction; seven with exercise intolerance and multiple deletions of mitochondrial DNA, presenting mild to moderate mitochondrial dysfunction; five without mitochondrial disease, as controls. Whole homogenates of muscle fragments were prepared using grinder-type equipment. O-2 consumption rates were normalized using citrate synthase activity. Results Transmission electron microscopy confirmed mitochondrial membrane discontinuation, indicating increased permeability of mitochondrial membranes in homogenates from frozen biopsies. O-2 consumption rates in the presence of acetyl-CoA lead to maximum respiratory rates sensitive to rotenone, malonate and antimycin. This protocol of acetyl-CoA-driven respiration (ACoAR), applied in whole homogenates of frozen muscle, was sensitive enough to identify ETC abnormality, even in patients with mild to moderate mitochondrial dysfunction. We demonstrated adequate repeatability of ACoAR and found significant correlation between O-2 consumption rates and enzyme activity assays of individual ETC complexes. Conclusions We present preliminary data on a simple, low cost and reliable procedure to measure respiratory function in whole homogenates of frozen skeletal muscle biopsies, contributing to diagnosis of mitochondrial diseases in humans. (AU)