高分辨率呼吸测量: 用于人体细胞和人体肌肉小活检的透化纤维的 OXPHOS 协议

High-resolution respirometry: OXPHOS protocols for human cells and permeabilized fibers from small biopsies of human muscle.

 

 

 

Keywords:Substrate–uncoupler–inhibitor titration , Human vastus lateralis , Needle biopsy , HEK ,HPMC , HUVEC , Fibroblasts , Routine respiration , Oxidative phosphorylation , Q-junction , Pyruvate ,
Glutamate , Malate , Succinate , Leak , Coupling control , Uncoupling , Oxygraph , Oxygen fl ux , Residual
oxygen consumption , Instrumental background 
关键词:底物-解偶联剂-抑制剂滴定,人股外侧肌,针活检,HEK,HPMC,HUVEC,成纤维细胞,常规呼吸,氧化磷酸化,Q-结,丙酮酸,谷氨酸 , 苹果 酸盐 , 琥珀酸盐 , 泄漏 , 耦合 控制 , 解 耦合 , 氧合仪 , 氧通量 , 残留. 耗氧量 , 仪器 背景 ;

细胞系:人脐静脉内皮细胞(正常人脐带静脉内皮细胞)
作者:Pesta D, Gnaiger E
出版期刊:《Mitochondrial Bioenergetics》 2011/10/3


Abstract:

Protocols for high-resolution respirometry of living cells, permeabilized cells, and permeabilized muscle fibers offer sensitive diagnostic tests of integrated mitochondrial function using standard cell culture techniques and small needle biopsies of muscle. Substrate-uncoupler-inhibitor titration (SUIT) protocols for analysis of oxidative phosphorylation improve our understanding of mitochondrial respiratory control and the pathophysiology of mitochondrial diseases. Respiratory states are defined in functional terms to account for the network of metabolic interactions in complex SUIT protols with stepwise modulation of coupling and substrate control. A regulated degree of intrinsic uncoupling is a hallmark of oxidative phosphorylation, whereas pathological and toxicological dyscoupling is evaluated as a mitochondrial defect. The noncoupled state of maximum respiration is experimentally induced by titration of established uncouplers (FCCP, DNP), to decrease the proton gradient across the mitochondrial inner membrane and measure the capacity of the electron transfer-pathway (Electron transfer pathway, open-circuit operation of respiration). Intrinsic uncoupling and dyscoupling are evaluated as the flux control ratio between non-phosphorylating LEAK respiration (electron flow coupled to proton pumping to compensate for proton leaks) and ET-capacity. If OXPHOS-capacity (maximally ADP stimulated oxygen flux) is less than ET-capacity, the phosphorylation system contributes to flux control. Physiological NS-substrate combinations support maximum Electron transfer pathway and OXPHOS capacities, due to the additive effect of multiple electron supply pathways converging at the Q-junction. Substrate control with electron entry separately through Complex I (pyruvate&malate or glutamate&malate) or Complex II (succinate&rotenone) restricts ET-capacity and artificially enhances flux control upstream of the Q-cycle, providing diagnostic information on specific branches of the Electron transfer pathway. Oxygen levels are maintained above air saturation in protocols with permeabilized muscle fibers to avoid experimental oxygen limitation of respiration.
Standardized two-point calibration of the polarographic oxygen sensor (static sensor calibration), calibration of the sensor response time (dynamic sensor calibration), and evaluation of instrumental background oxygen flux (systemic flux compensation) provide the unique experimental basis for high accuracy of quantitative results and quality control in high-resolution respirometry.

 

文章摘要:

活细胞、透化细胞和透化肌纤维的高分辨率呼​​吸测定协议使用标准细胞培养技术和肌肉的小针活检提供综合线粒体功能的敏感诊断测试。用于分析氧化磷酸化的底物-解偶联剂-抑制剂滴定 (SUIT) 协议提高了我们对线粒体呼吸控制和线粒体疾病病理生理学的理解。呼吸状态以功能术语定义,以解释复杂 SUIT 协议中的代谢相互作用网络,并逐步调节耦合和底物控制。调节程度的内在解偶联是氧化磷酸化的标志,而病理和毒理学脱偶联被评估为线粒体缺陷。最大呼吸的非耦合状态是通过滴定已建立的解偶联剂(FCCP,DNP)来实验诱导的,以降低穿过线粒体内膜的质子梯度并测量电子转移通路的容量(电子转移通路,开路操作呼吸)。内在解耦合和反耦合被评估为非磷酸化泄漏呼吸(电子流耦合到质子泵以补偿质子泄漏)和 ET 容量之间的通量控制比。如果 OXPHOS 能力(最大 ADP 刺激的氧通量)小于 ET 能力,则磷酸化系统有助于通量控制。生理 NS 底物组合支持最大的电子转移途径和 OXPHOS 容量,这是由于在 Q 结处会聚的多个电子供应途径的累加效应。通过复合物 I(丙酮酸和苹果酸或谷氨酸和苹果酸)或复合物 II(琥珀酸和鱼藤酮)分别进行电子进入的底物控制限制了 ET 容量并人为地增强了 Q 循环上游的通量控制,从而提供了有关电子转移途径特定分支的诊断信息。在具有透化肌纤维的协议中,氧气水平保持在空气饱和度以上,以避免呼吸的实验性氧气限制。
极谱式氧传感器的标准化两点校准(静态传感器校准)、传感器响应时间校准(动态传感器校准)、仪器背景氧通量评估(系统通量补偿)为高精度定量提供了独特的实验基础高分辨率呼​​吸测量的结果和质量控制。

 


点击链接即可查看和下载文章:https://link.springer.com/protocol/10.1007/978-1-61779-382-0_3
文章题目、关键词与摘要译文仅用于参考。 

APPLICATION CASE

应用案例