Lens redox fluorometry: pyridine nucleotide fluorescence and analysis of diabetic lens

We performed both ex vivo and in vivo fluorometric analyses of pyridine nucleotides (PN) in rabbit and rat lenses. Rabbit lens PN fluorescence (99% NADH) was found to have an excitation maximum at 366 nm and an emission maximum at 462 nm (366:462). The only other fluorescent chromophore in that region of the spectrum has excitation and emission peaks at 328 and 460 nm, respectively. Anaerobic glycolysis in the lens was stimulated by KCN, a known inhibitor of mitochondrial respiration, after which a time-study of fluorescence intensities was performed. Over the course of a 3.5 hr period following treatment with KCN, the PN signal showed a statistically significant increase relative to that in the control lenses (those treated with KCl). while the 328:460 signal (which may be due to some protein involved in energy transfer with the PN) had a significantly greater decrease. We also found that fluorescence intensity of NADH in solution is linearly proportional to physiologic-range concentration. Moreover, there was a close correlation between fluorescence intensity of rat lens PN as measured on a specular microscope-coupled redox fluorometer capable of in vivo use, and the lens PN levels as determined by the analytical cycling assay technique. This fluorometer was then employed to assess the redox state in rats with streptozotocin-induced diabetes. The normalized ratio of PN to flavoproteins (Fp) in the lens epithelium increased from 0.96 +/- 0.12 in the normal state to 1.48 +/- 0.30 2 weeks after diabetes induction. In contrast, the ratio in the diabetic lens treated with an aldose reductase inhibitor, sorbinil, did not increase. The increase in the PN:Fp ratio therefore reflects activation of the polyol pathway and its associated metabolic activities, which results in an increase in the NADH:NAD ratio in the diabetic rat lenses. Our results indicate that the non-invasive, real-time method of redox fluorometry may be useful in the early detection and evaluation of cataracts and other disorders in lens metabolism, long before opacities occur. It can be used to monitor the disease process and evaluate the efficacy of such drugs as aldose reductase inhibitors on a biochemical level.