Cellular NADH and NADPH Conformation as a Real-Time Fluorescence-Based Metabolic Indicator under Pressurized Conditions

• Main Authors: Martin Heidelman, Bibek Dhakal, Millicent Gikunda, Kalinga Pavan Thushara Silva, Laxmi Risal, Andrew I. Rodriguez, Fumiyoshi Abe, Paul Urayama
• Format: Article
• Language: English
• Published: MDPI AG 2021-08-01
• Series: Molecules
• Subjects: yeast; hydrostatic pressure; autofluorescence; spectral phasor analysis; NADH and NADPH conformation
• Online Access: https://www.mdpi.com/1420-3049/26/16/5020

Cellular conformation of reduced pyridine nucleotides NADH and NADPH sensed using autofluorescence spectroscopy is presented as a real-time metabolic indicator under pressurized conditions. The approach provides information on the role of pressure in energy metabolism and antioxidant defense with applications in agriculture and food technologies. Here, we use spectral phasor analysis on UV-excited autofluorescence from <i>Saccharomyces cerevisiae</i> (baker’s yeast) to assess the involvement of one or multiple NADH- or NADPH-linked pathways based on the presence of two-component spectral behavior during a metabolic response. To demonstrate metabolic monitoring under pressure, we first present the autofluorescence response to cyanide (a respiratory inhibitor) at 32 MPa. Although ambient and high-pressure responses remain similar, pressure itself also induces a response that is consistent with a change in cellular redox state and ROS production. Next, as an example of an autofluorescence response altered by pressurization, we investigate the response to ethanol at ambient, 12 MPa, and 30 MPa pressure. Ethanol (another respiratory inhibitor) and cyanide induce similar responses at ambient pressure. The onset of non-two-component spectral behavior upon pressurization suggests a change in the mechanism of ethanol action. Overall, results point to new avenues of investigation in piezophysiology by providing a way of visualizing metabolism and mitochondrial function under pressurized conditions.