MITOCHONDRIAL RESPIROMETRY IN FROZEN BIOLOGICAL SAMPLES

冷冻生物样品中的线粒体呼吸测定

• 批准号: 10251412
• 负责人: Orian S Shirihai
• 金额: $ 7.67万
• 依托单位: ENSPIRE BIO, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-26 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10251412
• 关键词: ATP Hydrolysis; ATP Synthesis Pathway; Adult; Aging; Biological; Biological Assay; Cardiovascular system; Cell Line; Clinical; Clinical Research; Communities; Complex; Data; Diagnostic; Disease; Electron Transport; Freezing; Functional disorder; Goals; Heart; Heart Mitochondria; Hour; Hydrolysis; Impairment; Inner mitochondrial membrane; Legal patent; Literature; Liver Mitochondria; Measurement; Measures; Membrane Potentials; Metabolic; Mitochondria; Mitochondrial DNA; Mus; Mutation; Oxygen Consumption; Patients; Play; Polymerase; Protocols documentation; Publishing; Reading; Respiration; Respiratory physiology; Retrospective Studies; Rodent; Sampling; Side; Techniques; Technology; Testing; Time; Tissue Sample; Tissues; aged; clinical application; clinical practice; cost; improved; mitochondrial membrane; mouse model; new technology; novel; oligomycin sensitivity-conferring protein; respiratory; tool

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.

抽象的 线粒体呼吸受损在代谢，心血管和衰老有关的关键作用 疾病。但是，线粒体呼吸测定法当前需要处理活组织 从患者带走后一个小时内采样。该要求使呼吸测定法分析很大程度上 对标准临床实践和临床研究不可行。我们发明了一项新技术来评估最大 以前冷冻的生物样品中的线粒体呼吸能力，到目前为止被认为 科学界不可能。并排比较证实了我们的测定准确反映 结果在新鲜样品中测量。在此之前，我们还没有考虑测量复杂V的技术 由于我们知道以前冷冻的线粒体由于内部的破坏而不会合成ATP 线粒体膜和线粒体膜电位的损失。 Enspire Bio将进一步开发这种专利的冷冻丝图技术，以实现呼吸测量 临床应用和冷冻样品的ATP合酶（复杂V）活性，目前不是 可用的。我们将通过AIM 1：为ATP合酶复合物v建立标准协议来实现我们的目标 从衍生的冷冻组织样品中，已经存储了细胞系和小鼠组织样品。我们将进一步改善 通过启用从组织匀浆的氧气消耗而不是分离的吞吐量 线粒体。这种方法允许回顾性研究检查线粒体功能的变化和 在最小和非侵入性临床样品中具有潜在利用作为诊断工具。而这种技术 可用于确定电子传输链复合物I，II和IV的变化，我们尚未 评估了先前冷冻样品中的线粒体复合物V。