Measurement of mitochondrial NADH and FAD autofluorescence in live cells.
Measurement of mitochondrial NADH and FAD autofluorescence in live cells.
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DOI：
10.1007/978-1-4939-2257-4_23
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发表时间：
2015-01-01
期刊：
影响因子：
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通讯作者：
Abramov, Andrey Y
中科院分区：
文献类型：
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作者：
Bartolome, Fernando;Abramov, Andrey Y
在能量产生过程中，线粒体网络是底物代谢为 ATP 的关键要素。许多病理状况与线粒体功能障碍有关，因为线粒体与多种细胞过程相关。因此，能量产生的任何破坏都会引起破坏性影响，最终导致细胞因化学缺血而死亡。为了解决线粒体的健康和功能问题，有几个反映整个线粒体功能或单个线粒体复合物的生物能参数。特别是，三羧酸循环中营养物质的代谢提供了用于产生电子载体（烟酰胺腺嘌呤二核苷酸[NADH]和黄素腺嘌呤二核苷酸[FADH2]）的底物，最终将电子贡献给线粒体电子传递链。 NADH 和 FADH2 的水平可以通过 NADH/NAD(P)H 或 FAD 自发荧光成像来估计。该报告演示了如何以时间过程依赖性方式执行和分析 NADH/NAD(P)H 和 FAD 自发荧光，并提供有关 NADH 和 FAD 氧化还原指数的信息，两者均反映线粒体电子传递链 (ETC) 的活性。此外，还可以估算 NADH 和 FAD 的总池量，从而提供有关 ETC 底物供应速率的信息。最后，诱导最大呼吸后 NADH 自发荧光的分析可以提供有关磷酸戊糖途径活性的信息，其中葡萄糖可以替代氧化而不是丙酮酸。
In the process of energy production, mitochondrial networks are key elements to allow metabolism of substrates into ATP. Many pathological conditions have been associated with mitochondrial dysfunction as mitochondria are associated with a wide range of cellular processes. Therefore, any disruption in the energy production induces devastating effects that can ultimately lead to cell death due to chemical ischemia. To address the mitochondrial health and function, there are several bioenergetic parameters reflecting either whole mitochondrial functionality or individual mitochondrial complexes. Particularly, metabolism of nutrients in the tricarboxylic acid cycle provides substrates used to generate electron carriers (nicotinamide adenine dinucleotide [NADH] and flavin adenine dinucleotide [FADH2]) which ultimately donate electrons to the mitochondrial electron transport chain. The levels of NADH and FADH2 can be estimated through imaging of NADH/NAD(P)H or FAD autofluorescence. This report demonstrates how to perform and analyze NADH/NAD(P)H and FAD autofluorescence in a time-course-dependent manner and provides information about NADH and FAD redox indexes both reflecting the activity of the mitochondrial electron transport chain (ETC). Furthermore, total pools of NADH and FAD can be estimated providing information about the rate of substrate supply into the ETC. Finally, the analysis of NADH autofluorescence after induction of maximal respiration can offer information about the pentose phosphate pathway activity where glucose can be alternatively oxidized instead of pyruvate.