Measurement of fluorescein and fluorescein monoglucuronide in the living human eye.

Fluorescein monoglucuronide is a fluorescent metabolite of fluorescein, and is 1/3 to 1/34 as fluorescent as fluorescein, depending on the wavelength of excitation. After systemic administration, fluorescein glucuronide reaches concentrations many times greater than fluorescein. In order to study the effect of fluorescein glucuronide on the measurement of ocular dynamics, we devised a technique to measure fluorescein and fluorescein glucuronide in the anterior segment of the living human eye. Concentrations of each fluorophore were determined by differential spectrofluorophotometry from measurements at excitation wavelengths of 457.9 nm and 488.0 nm. Measurements were made on normal volunteers after oral and intravenous administration of fluorescein. Fluorescein was the dominant fluorophore during the first hour, while fluorescein glucuronide became dominant after 3 hours. By 6 hours there was 10 to 30 times more fluorescein glucuronide than fluorescein in the anterior chamber after oral administration, and three to ten times more after intravenous administration. The blood aqueous diffusion coefficient kd estimated from the apparent concentration of fluorescein measured at 457.9 nm was consistently greater than kd estimated from measurements at 488.0 nm. Estimates of kd, which were made on the basis of concentrations of fluorescein determined from measurements at both wavelengths, were lower than estimates based on measurements at either wavelength. These results indicate that wavelength of excitation may influence the determination of ocular parameters when systemic fluorescein is used. Care must be taken in the interpretation of measurements when metabolites of a fluorophore can interfere with measurement of the fluorophore itself.