Autofluorescence of the diabetic and healthy human cornea in vivo at different excitation wavelengths.
Corneal autofluorescence is higher in diabetes mellitus patients with retinopathy than in healthy subjects. In this study, the excitation spectra of corneal autofluorescence of diabetic patients and healthy controls in the range 365 nm-480 nm were compared in an attempt to identify the fluorophores responsible for corneal autofluorescence in health and disease (diabetes). Spectral measurements (from one eye) were recorded from five patients with proliferative diabetic retinopathy and five age-matched healthy controls, using a modified commercial scanning fluorophotometer with a mercury arc or a tungsten halogen lamp as excitation light source in combination with interference filters (excitation wavelengths: 365, 405, 420, 430, 436, 440, 450, 470 and 480 nm; bandwidth: 10 nm). Fluorescence emission was measured in the range 532 nm-630 nm. The sensitivity of the modified fluorophotometer was calibrated by using the excitation spectrum of fluorescein as a reference. The corneal excitation efficiency of the diabetic patients was higher than that of the healthy controls at each wavelength investigated (Mann-Witney test P<0.0005). The ratio between the mean values of both groups was equal for each excitation wavelength (mean ratio 1.9+/-0.12s.d.,P>0. 2), suggesting that the excitation spectra were equal. This indicates that the same fluorophores are responsible for the corneal autofluorescence in both groups. The shapes of the excitation spectra suggest the involvement of flavins, NAD(P)H, and at least one other, as yet unidentified, fluorophore.