Assessment of Cellular Redox State Using NAD(P)H Fluorescence Intensity and Lifetime   

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A brief version of this protocol appeared in:
EMBO Molecular Medicine
May 2016


NADH and NADPH are redox cofactors, primarily involved in catabolic and anabolic metabolic processes respectively. In addition, NADPH plays an important role in cellular antioxidant defence. In live cells and tissues, the intensity of their spectrally-identical autofluorescence, termed NAD(P)H, can be used to probe the mitochondrial redox state, while their distinct enzyme-binding characteristics can be used to separate their relative contributions to the total NAD(P)H intensity using fluorescence lifetime imaging microscopy (FLIM). These protocols allow differences in metabolism to be detected between cell types and altered physiological and pathological states.

Keywords: NADH, NADPH, NAD(P)H, Autofluorescence, Microscopy, Fluorescence lifetime, FLIM, Redox state


The reduced form of the redox cofactor nicotinamide adenine dinucleotide (NADH) and its phosphorylated counterpart NADPH are intrinsically fluorescent, both absorbing light at wavelengths of 340 (± 30) nm and emitting at 460 (± 50) nm (Patterson et al., 2000). These spectral characteristics are lost upon oxidation to NAD+ or NADP+ (De Ruyck et al., 2007). The redox balances of the separate NAD and NADP pools dictate contrasting metabolic processes (Ying, 2008), as shown in Figure 1. NAD acts as an electron acceptor for the oxidation of sugar, lipid and amino acid substrates in the mitochondria by the tricarboxylic acid (TCA) cycle and as an electron donor to the electron transport chain (ETC) on the inner mitochondrial membrane (IMM), fuelling the pumping of protons into the intermembrane space to act as a power source for the synthesis of adenosine triphosphate (ATP) by the F1FO ATP synthase (Osellame et al., 2012). The balance of NADH to NAD+ in the mitochondria therefore reflects the balance of TCA cycle to ETC activity. ETC dysfunction causes increases in the NADH/NAD+ ratio and the production of potentially damaging reactive oxygen species (ROS) (Murphy, 2009). The cell’s antioxidant defences require the NADP pool to provide reducing equivalents for their maintenance, so the NADPH/NADP+ ratio must be maintained high (> 3) (Pollak et al., 2007). The redox state of the mitochondrial NAD pool and the relative abundance of NADPH are therefore key factors in the level of oxidative stress in a cell type.

Figure 1. Schematic outline of mitochondrial NAD(P)H metabolism. Substrate oxidation in the TCA cycle passes electrons to NAD+, forming NADH. A. Under resting conditions, electrons carried by NADH are passed along the ETC, powering the pumping of protons from the mitochondrial matrix across the inner mitochondrial membrane (IMM) into the intermembrane space. The resulting proton gradient powers the production of ATP at complex V of the ETC (F1FO ATP synthase). B. Addition of an uncoupler such as carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) allows protons to leak back across the IMM, causing the rate of oxidation of NADH at the ETC to increase to restore the membrane gradient. C. Inhibition of the ETC by rotenone halts NADH oxidation and causes an increase in the production of superoxide (O2-), the proximal source of mitochondrial ROS. This is neutralised upon its conversion into water by the superoxide dismutase (SOD2) and glutathione (GSH/GSSG) antioxidant defence systems, maintained by NADPH.

Here, we describe protocols for the assessment of the mitochondrial NADH/NAD+ ratio and the NADPH/NADH balance that rely on the fluorescence of these cofactors when reduced. Their identical absorption and emission spectra leads the combined signal to be termed NAD(P)H (Blacker and Duchen, 2016). Measuring the change in NAD(P)H fluorescence using a confocal microscope following the application of an ETC uncoupler and inhibitor allows the mitochondrial NADH/NAD+ balance to be estimated (Duchen et al., 2003). To discriminate between the relative contributions of NADH and NADPH to the total signal, fluorescence lifetime imaging microscopy (FLIM) must be introduced (Blacker et al., 2014). These protocols describe, in further detail, methods used in Tosatto et al. (2016) to investigate the role of the selective channel responsible for mitochondrial calcium uptake, the mitochondrial calcium uniporter (MCU), in the progression of breast cancer. Basic understanding of confocal microscopy is assumed. For background, readers are directed to Pawley et al. (2012).

Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Blacker, T. S., Berecz, T., Duchen, M. R. and Szabadkai, G. (2017). Assessment of Cellular Redox State Using NAD(P)H Fluorescence Intensity and Lifetime. Bio-protocol 7(2): e2105. DOI: 10.21769/BioProtoc.2105.

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