Noninvasive measurements of pyridine nucleotide fluorescence from the cornea

The autofluorescence of reduced pyridine nucleotides (NADH and NADPH) and oxidized flavoproteins within the rabbit cornea were noninvasively measured as a function of depth. This was accomplished by combining a corneal specular microscope with a time-shared spectrofluorometer. When either 8 mM sodium pentobarbital or sodium sulfide, known inhibitors of mitochondrial respiration were applied to cornea, the autofluorescence at 440 nm (excited at 366 nm) increased and that at 540 nm (excited at 460 nm) decreased. No autofluorescence was measurable following destruction of the cellular membranes by freezing and leaching of the cellular constituents. The 440 nm autofluorescence is from reduced pyridine nucleotides, whereas the 540 nm autofluorescence is from the oxidized flavoproteins. The time course of the pyridine nucleotide autofluorescence after the application of the pentobarbital to either the endothelial or epithelial bathing solutions made it possible to measure the diffusion properties of this drug through the cornea. The method used is useful studying the diffusion and effects of metabolically active drugs upon the cornea.