Objectives of Study

It is a major complication of the non-invasive study of the blood-retina barrier that the commonly used tracer cannot easily be distinguished from its principal metabolite. Fluorescein, the only tracer presently approved for use in human subjects, is rapidly converted to fluorescein monoglucuronide, which has excitation and fluorescence spectra that are roughly comparable to those of fluorescein. A further difficulty is that the fluorophores in the vitreous must be detected through colored, semi-opaque refractive media, the optical densities of which are known to vary with age and disease. The objective of the studies described in this thesis was to develop improved methods for the non-invasive fluorometric assessment of the lens in order to enable the separate determination of fluorescein and fluorescein glucuronide in the vitreous and the bloodretina barrier permeabilities of these two fluorophores. The methodological developments were also applied to the study of other fundamental aspects of ocular pathophysiology in diabetes mellitus. A prerequisite for the quantitative analysis of ocular fluorescence is that the exact location of the fluorescence measurement is known. This was determined by analysing the geometrical optics of the ocular fluorometer and the eye and by developing an experimentally based model of the two systems. A new principle was developed for the analysis of intrinsic lens fluorescence and for the estimation of lens transmittance. The method was validated by in vitro experiments. To improve our understanding of the relation between systemic pathophysiological conditions and the lens, fluorometric studies were made of lens changes in diabetic patients in relation to metabolic control and kidney function. The fluorescence of fluorescein and fluorescein glucuronide was examined in the vitreous in vitro to determine if the fluorescence in this medium is comparable to that in standard reference solutions. The plasma pharmacokinetics of the two fluorophores were studied to determine whether the interindividual variation in a relevant patient group is large enough to warrant plasma sampling and analysis in every single patient undergoing vitreous fluorometry . An ocular fluorometer was rebuilt for in vivo detection of fluorescein and fluorescein glucuronide. On the basis of analytical principles developed in the prior studies the blood-retina barrier leakage of fluorescein and fluorescein glucuronide was examined in insulin-dependent diabetic patients with background retinopathy.