In Vivo Analysis of Hepatic NADH Fluorescence

Abstract

Direct observation of intracellular events and their relationship to physiological function presents a challenging and long-standing problem. The determination of intracellular oxygen levels in tissues has been a subject of continuous discussion, but most of the techniques for measuring oxygen tension in blood and tissue fail to indicate the oxidationreduction states of the respiratory carriers. Observations of the latter, however, would be of particular importance, since they reflect the intracellular level of energy transfering components. Reduced nicotinamide adenine dinucleotide (NADH) is one of the main means of energy transfer from the tricarboxylic acid cycle to the respiratory chain in the mitochondria. Most of the energy derived from aerobic metabolism is initially generated as NADH, which is oxidized to NAD⁺ as electrons are passed to the electron transport chain and, ultimately, to molecular oxygen. Decades ago, Chance and coworkers pioneered the fluorescence property of NADH as an indicator of the mitochondrial redox state and, in the presence of sufficient substrate and phosphate, as an indicator of cellular oxygen (Chance et al., 1962; Chance, 1976). For example, interruption of oxidative phosphorylation is associated with a prompt increase of tissue NADH levels, and that increase can be monitored by following its optical fluorescence (Chance and Schoener, 1965; Chance et al., 1965).The optical properties of NADH and NAD⁺ clearly differ in that upon ultraviolet excitation (330–390 nm), NADH, unlike NAD⁺, fluoresces in the blue (>430 nm). Meanwhile, NADH fluorescence measurements have been applied to single cells, cell suspensions, tissue slices, ex vivo perfused organs, but also to organs in vivo (Ince et al., 1992).