Department of Neurosciences, Division of Neurology, Ribeirão Preto Medical School, University of São Paulo, São Paulo, Brazil.
Department of Cell and Molecular Biology, Ribeirão Preto Medical School, University of São Paulo, São Paulo, Brazil.
Eur J Clin Invest. 2021 Sep;51(9):e13574. doi: 10.1111/eci.13574. Epub 2021 May 2.
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.
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 consumption rates were normalized using citrate synthase activity.
Transmission electron microscopy confirmed mitochondrial membrane discontinuation, indicating increased permeability of mitochondrial membranes in homogenates from frozen biopsies. O 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 consumption rates and enzyme activity assays of individual ETC complexes.
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.
在临床实践中，冷冻人类活检组织用于存储是很常见的。然而，这种技术会破坏线粒体膜，妨碍进一步分析呼吸功能。为了有助于实验室诊断线粒体疾病，本研究旨在开发一种使用 O2k（Oroboros Ins.）的呼吸测量方法，以测量冷冻骨骼肌活检匀浆中的整个电子传递链（ETC）活性。
我们招募了 16 名在常规诊断研究过程中接受肌肉活检的患者：4 名患有线粒体疾病和严重线粒体功能障碍；7 名运动不耐受和线粒体 DNA 多处缺失，表现为轻度至中度线粒体功能障碍；5 名无线粒体疾病作为对照。使用研磨器类型的设备制备肌肉片段的全匀浆。使用柠檬酸合酶活性对 O 消耗率进行归一化。
透射电子显微镜证实了线粒体膜的中断，表明冷冻活检匀浆中线粒体膜的通透性增加。在乙酰辅酶 A 存在下的 O 消耗率导致对鱼藤酮、丙二酸和抗霉素敏感的最大呼吸速率。该乙酰辅酶 A 驱动的呼吸（ACoAR）方案，应用于冷冻肌肉的全匀浆中，足以识别 ETC 异常，即使在轻度至中度线粒体功能障碍的患者中也是如此。我们证明了 ACoAR 的可重复性足够好，并发现 O 消耗率与个体 ETC 复合物的酶活性测定之间存在显著相关性。
我们提出了一种简单、低成本和可靠的程序，用于测量冷冻骨骼肌活检全匀浆中的呼吸功能，有助于人类线粒体疾病的诊断。
