MITOCHONDRIAL RESPIROMETRY IN FROZEN BIOLOGICAL SAMPLES

摘要 线粒体呼吸受损在代谢、心血管和衰老相关疾病中起着关键作用 疾病。然而，线粒体呼吸测量分析目前需要对活组织进行处理 在从病人身上取下样本后一小时内。这一要求使得呼吸测量分析变得非常重要 对于标准的临床实践和临床研究是不可行的。我们发明了一种新技术来评估最大 之前冷冻的生物样本中的线粒体呼吸能力，到目前为止还被认为是 科学界认为这是不可能的。并列比对证实了我们的分析准确地反映了 结果在新鲜样品中测量。直到现在，我们还没有考虑过测量复数V的技术 因为我们知道之前冻结的线粒体由于内部的破坏而不能合成ATP 线粒体膜和线粒体膜电位丧失。 Enspire Bio将进一步开发这项获得专利的冷冻Mitos技术，以实现呼吸测量 以及用于临床应用和冷冻标本的ATP合成酶(复合体V)活性，这目前不是 可用。我们将通过目标1：建立ATP合成酶复合体V的标准方案来实现我们的目标 从冷冻的组织样本中提取已经储存的细胞系和小鼠组织样本。我们将进一步完善 通过测量组织匀浆的耗氧量而不是孤立的 线粒体。这种方法允许进行回溯性研究，以检查线粒体功能和 在微创和非侵入性临床样本中具有潜在的诊断工具的应用。虽然这项技术 可以用来测定电子传递链复合体I、II和IV的变化，我们还没有 评估了先前冷冻样本中的线粒体复合体V。

Abstract Impaired mitochondrial respiration plays a key role in metabolic, cardiovascular, and aging-related diseases. However, mitochondrial respirometry analysis currently requires processing of the living tissue sample within an hour after being taken from the patient. This requirement makes respirometry analysis largely unfeasible to standard clinical practice and clinical studies. We invented a new technology to assess maximal mitochondrial respiratory capacity in previously-frozen biological samples, which thus far been considered impossible by the scientific community. Side-by-side comparison confirmed that our assay accurately reflects results measured in fresh samples. Until this point, we had not considered techniques to measure Complex V since we know that previously frozen mitochondria do not synthesize ATP due to disruption in the inner mitochondrial membrane and loss of mitochondrial membrane potential. Enspire Bio will further develop this patented Frozen Mitos technology to enable respirometry measurements and ATP synthase (Complex V) activity for clinical applications and frozen samples, which currently is not available. We will achieve our goals by Aim 1: Establishing standard protocols for ATP synthase Complex V from frozen tissue samples derived already stored cell lines and mouse tissue samples. We will further improve throughput by enabling measurements of oxygen consumption from tissue homogenate instead of isolated mitochondria. This approach allows for retrospective studies to examine changes in mitochondrial function and has potential utilization as a diagnostic tool in minimally and non-invasive clinical samples. While this technique can be used to determine changes in electron transport chain Complexes I, II, and IV, we have not yet assessed the mitochondrial Complex V in previously frozen samples.