Investigating mitochondrial redox state using NADH and NADPH autofluorescence

Abstract

The redox states of the NAD and NADP pyridine nucleotide pools play critical roles in defining the activity of energy producing pathways, in driving oxidative stress and in maintaining antioxidant defences. Broadly speaking, NAD is primarily engaged in regulating energy-producing catabolic processes, whilst NADP may be involved in both antioxidant defence and free radical generation. Defects in the balance of these pathways are associated with numerous diseases, from diabetes and neurodegenerative disease to heart disease and cancer. As such, a method to assess the abundance and redox state of these separate pools in living tissues would provide invaluable insight into the underlying pathophysiology. Experimentally, the intrinsic fluorescence of the reduced forms of both redox cofactors, NADH and NADPH, has been used for this purpose since the mid-twentieth century. In this review, we outline the modern implementation of these techniques for studying mitochondrial redox state in complex tissue preparations. As the fluorescence spectra of NADH and NADPH are indistinguishable, interpreting the signals resulting from their combined fluorescence, often labelled NAD(P)H, can be complex. We therefore discuss recent studies using fluorescence lifetime imaging microscopy (FLIM) which offer the potential to discriminate between the two separate pools. This technique provides increased metabolic information from cellular autofluorescence in biomedical investigations, offering biochemical insights into the changes in time-resolved NAD(P)H fluorescence signals observed in diseased tissues.

Lactate is oxidized outside of the mitochondrial matrix in rodent brain

“…Though previous studies have demonstrated that the majority of cellular NAD + (>75%) is located inside the mitochondria (Tischler et al 1977;Di Lisa and Ziegler 2001), the cytosolic NAD + /NADH redox potential is reported to greatly exceed the mitochondrial NAD + / NADH potential (Williamson et al 1967;Jones and Sies 2015;see Sahlin et al 2002 for discussion), suggesting that lactate oxidation is favoured outside of the mitochondrial matrix. In the cytosol, this is due to the equilibrium constant for the associated redox couples (namely, lactate/pyruvate) combined with influential protein binding sites for NADH (Jones and Sies 2015); changes in NADH are therefore primarily mitochondrial (Sun et al 2012;Blacker and Duchen 2016). Indeed, consistent with the present study, lactate oxidation only occurs when NAD + is added.…”

Autofluorescence lifetime imaging of cellular metabolism: Sensitivity toward cell density, pH, intracellular, and intercellular heterogeneity

“…The effect of pH on NADH based FLIM has been performed before to understand the stress response and involvement of NADPH production in related to the ROS(reactive oxygen species) production(57,58). Although there has been report of FLIM-NADPH response with ROS(59), currently there are no accurate ways to robustly characterize this response. The intensity drop between time 2.4 hours and 6.6 hours shows possible photobleaching over time or couple of apoptotic cells which reduced the number of cells in the image over time.…”

NADH Autofluorescence—A Marker on its Way to Boost Bioenergetic Research

“…Consequently one has to be aware of the fact that the measured autofluorescence is always a mixture of both NADPH and NADH (33). Consequently one has to be aware of the fact that the measured autofluorescence is always a mixture of both NADPH and NADH (33).…”