|Zuccolotto-dos-Reis FH, Andriao-Escarso SH, Araujo JS, Espreafico EM, Alberici LC, Sobreira CFR (2021) Acetyl CoA driven respiration in frozen muscle contributes to the diagnosis of mitochondrial disease. MitoFit Preprints 2021.3. doi:10.26124/mitofit:2021-0003 - 2021-09 Published in »European Journal of Clinical Investigation 51(9):e13574«|
Abstract: Version 1 (v1) 2021-02-19 doi:10.26124/mitofit:2021-0003 - 2021-09 Published in »European Journal of Clinical Investigation 51(9):e13574«
BACKGROUND: the procedure of freezing human biopsies is common in clinical practice as a form of storage. However, this technique disrupts mitochondrial membranes, hampering further analyses of respiratory function. To contribute to the laboratorial diagnosis of mitochondrial diseases, this study sought to develop an O2 consumption protocol to measure the whole electron transfer system (ETS) 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; and five without mitochondrial disease, as controls. Whole homogenates of muscle fragments were prepared using grinder-type equipment. RESULTS: Transmission electron microscopy confirmed that most mitochondria presented areas of membrane discontinuation, indicating increased permeability of mitochondrial membranes in homogenates from frozen biopsies. O2 consumption rates in the presence 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 ETS abnormality, even in patients with mild to moderate mitochondrial dysfunction. We demonstrated adequate repeatability of ACoAR and found a significant correlation between O2 consumption rates and enzyme activity assays of individual ETS complexes. CONCLUSIONS: here we present a simple, low cost and reliable procedure to measure respiratory function in whole homogenates of frozen skeletal muscle biopsies, contributing to the diagnosis of mitochondrial diseases in humans.
• Keywords: frozen skeletal muscle biopsy, acetyl-CoA driven respiration (ACoAR), oxygen consumption rate, high-resolution respirometry, electron transfer system, mitochondrial diseases • Bioblast editor: Cardoso LHD • O2k-Network Lab: BR Ribeirao Preto Alberici LC
- As editors, it is not our responsibility to review the scientific content of a manuscript submitted to MitoFit Preprints. As scientists discussing a publication, this is our routine task.
- A critical assessment of fresh homogenates is missing. How does fresh homogenate (used as a reference for cryopreservation) compare to state-of-the-art mitochondrial preparations of (human) skeletal muscle tissue? Even without such additional methodological experiments, there is a huge database available in the literature for comparison. The comparison indicates mitochondrial damage even in the fresh homogenates.
- The information content of ‘basal’ and ‘AcCoA activated’ respiratory activity (normalized for CS) in homogenates of cryopreserved samples needs to be evaluated relative to (1) conventional enzyme activity measurements, and (2) OXPHOS analysis in preparations of functional mitochondria.
Labels: MiParea: Respiration, Instruments;methods