Accuracy of aqueous humor flow determination by fluorophotometry

After corneal iontophoresis of fluorescein, freshly enucleated rhesus monkey eyes were perfused at a known rate with glutathione-bicarbonate-Ringer's solution. Total fluorescein mass and aqueous fluorescein concentration were measured with a fluorophotometer every 15 min for 6 to 8 hr. The aqueous flow was calculated from the fluorophotometry data by the second method of Jones and Maurice. This flow was then compared to the known perfusion rate. The curve for total fluorescence decay was not precisely parallel to the curve for aqueous fluorescence decay. This affected the value of the ratio of total:aqueous fluorescence. The most accurate and consistent flow values were obtained with the decay constant from the aqueous fluorescence decay curve combined with the ratio of total:aqueous fluorescence extrapolated back to zero time. Under these two conditions, the calculated flow in five experiments was 5.4% +/- 3.3 (+/- S.D.) lower than the known flow. The 99% confidence interval of the true mean percent difference between the known and calculated flows is--12% to +1%. A small effect of corneal fluorescein concentration on measured aqueous fluorescence was observed.