Distinguishing chemically induced NADPH- and NADH-related metabolic responses using phasor analysis of UV-excited autofluorescence.

NADPH and NADH are well known for their role in antioxidant defense and energy metabolism, respectively, however distinguishing their cellular autofluorescence signals is a challenge due to their nearly identical optical properties. Recent studies applying spectral phasor analysis to autofluorescence emission during chemically induced metabolic responses showed that two-component spectral behavior, i.e., spectral change acting as a superposition of two spectra, depended on whether one or multiple metabolic pathways were affected. Here, we use this property of spectral behavior to show that metabolic responses primarily involving NADPH or NADH can be distinguished. We start by observing that the cyanide-induced response at micro- and millimolar concentrations does not follow mutual two-component spectral behavior, suggesting their response mechanisms differ. While respiratory inhibition at millimolar cyanide concentration is well known and associated with the NADH pool, we find the autofluorescence response at micromolar cyanide concentration exhibits two-component spectral behavior with NADPH-linked EGCG- and peroxide-induced responses, suggesting an association with the NADPH pool. What emerges is a spectral phasor map useful for distinguishing cellular autofluorescence responses related to oxidative stress versus cellular respiration.

A metabolic interpretation for the response of cellular autofluorescence to chemical perturbations assessed using spectral phasor analysis.

Analytical approaches for sensing cellular NADH conformation from autofluorescence signals have significance because NADH is a metabolic indicator and endogenous biomarker. Recently, spectral detection of multiple cellular NADH forms during chemically-induced metabolic response was reported, however because NADH is solvatochromic and the spectral change is small, the possibility of a non-metabolic interpretation needs to be considered. Here we investigate the response of UV-excited autofluorescence to a range of well-known chemicals affecting fermentation, respiration, and oxidative-stress pathways in Saccharomyces cerevisiae. The two-component nature of the spectral response is assessed using phasor analysis. By considering a series of physically similar and dissimilar chemicals acting on multiple pathways, we show how the two-component nature of a spectral response is of metabolic origin, indicative of whether a single or several pathways have been affected.