Department of Medicine, Endocrinology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA.
Metabolism Theme, David Geffen School of Medicine, University of California, Los Angeles, CA, USA.
EMBO J. 2020 Jul 1;39(13):e104073. doi: 10.15252/embj.2019104073. Epub 2020 May 20.
Respirometry is the gold standard measurement of mitochondrial oxidative function, as it reflects the activity of the electron transport chain complexes working together. However, the requirement for freshly isolated mitochondria hinders the feasibility of respirometry in multi-site clinical studies and retrospective studies. Here, we describe a novel respirometry approach suited for frozen samples by restoring electron transfer components lost during freeze/thaw and correcting for variable permeabilization of mitochondrial membranes. This approach preserves 90-95% of the maximal respiratory capacity in frozen samples and can be applied to isolated mitochondria, permeabilized cells, and tissue homogenates with high sensitivity. We find that primary changes in mitochondrial function, detected in fresh tissue, are preserved in frozen samples years after collection. This approach will enable analysis of the integrated function of mitochondrial Complexes I to IV in one measurement, collected at remote sites or retrospectively in samples residing in tissue biobanks.
呼吸测量法是测量线粒体氧化功能的金标准，因为它反映了一起协同工作的电子传递链复合物的活性。然而，对新鲜分离的线粒体的需求阻碍了呼吸测量法在多地点临床研究和回顾性研究中的可行性。在这里，我们描述了一种新颖的呼吸测量法，通过恢复冷冻/解冻过程中丢失的电子传递成分并校正线粒体膜的通透性变化，适用于冷冻样本。该方法在冷冻样本中保留了 90-95%的最大呼吸能力，并且可以应用于具有高灵敏度的分离线粒体、透化细胞和组织匀浆。我们发现，在新鲜组织中检测到的线粒体功能的原发性变化在采集多年后仍保存在冷冻样本中。这种方法将能够在一次测量中分析线粒体复合物 I 到 IV 的综合功能，这些测量可以在远程地点或在组织生物库中存放的样本中进行回顾性分析。
