Plasma fluorescein and fluorescein glucuronide in patients with selected eye diseases.

Systemically administered fluorescein (F) is rapidly transformed to the fluorescent metabolite fluorescein glucuronide (FG). Little is known about how diseases can influence the synthesis or disposition of FG. We studied F and FG in the plasma ultrafiltrate of 75 people who were normal or had diabetes, retinitis pigmentosa, or idiopathic rhegmatogenous retinal detachment. F and FG were determined by high-performance liquid chromatography. The concentration of FG was comparable to F 1 h after an intravenous injection of F, both in normal subjects and in patients with retinitis pigmentosa, which suggests that FG may not be an important contributor to the vitreous fluorescence at that time. At later times FG substantially exceeded F. The concentration of FG was significantly higher in diabetics than in the other groups 14 h after an oral dose of F. Accordingly, the possible effect of disease on plasma dye concentrations should be considered in studies measuring F by fluorescence hours after systemic F administration, since this could influence the intraocular fluorescence irrespective of any alteration in ocular function.

Blood retinal and blood aqueous barriers in diabetics by fluorophotometry.

The permeability of the blood retinal barrier (PBRB) and the diffusion coefficient into the anterior chamber (kd) in 20 insulin dependent (ID) and in 11 non-insulin dependent (NID) diabetics with various degrees of retinopathy were determined by fluorophotometry with a 25% accuracy. The difference of PBRB and the kd values between the NID and the ID patients was not significant (p greater than 0.05 and p greater than 0.22). The mean PBRB and the mean kd values differed significantly from those of a healthy population (p less than 0.0013 and p = 0.025). A significant correlation was established between PBRB and diabetes duration (r = 0.55; p less than 0.01) but not between PBRB or kd values and metabolic control (HbAl) (p greater than 0.5) or creatinine clearance (p greater than 0.5). The time integrals of unbound plasma fluorescein in the diabetics between the time of injection and 1 hour later were comparable with those of healthy controls. The difference between the mean PBRB value of diabetics with advanced retinopathy and that of a healthy population was significant (p less than 0.003) but the difference for the mean values of diabetics with no or minimal background retinopathy was not (p greater than 0.2), indicating that PBRB values do not increase to an abnormal level while signs of diabetic retinopathy are absent on fluorescein angiography.

Diffusion of fluorescein-labelled molecules in suspensions of erythrocyte ghosts.

The diffusion of fluorescein isothiocyanate-labelled dextran molecules in suspensions of centrifugally, tightly packed, erythrocyte ghosts was measured by fluorescence recovery after photobleaching. In comparison with diffusion in aqueous solution, the diffusion coefficients for probe molecules of varying size were about two orders of magnitude smaller. It was established that the dextran molecules remained in the space between the ghosts. Since crosslinking membrane surface carbohydrates with antibodies further inhibits diffusion, it is assumed that interactions between surface carbohydrates and the probe molecules are the cause of slow diffusion. Two alternative models are discussed.

Carboxyfluorescein distribution in ocular tissues of normal and diabetic rats.

Quantitative fluorescence microscopy was used to study carboxyfluorescein distribution across the blood-ocular barrier of control and streptozotocin diabetic rats at 2 min, 1 and 2 hr after dye injection (125 mg/kg iv). Measurement of dye concentrations in plasma, urine and feces demonstrated increased plasma clearance and increased urinary clearance of carboxyfluorescein in diabetic rats. Unbound plasma dye concentration in the diabetic animals fell to 34% of the control level at 1 hr after injection; the corresponding plasma concentration vs. time integral was reduced to only 74% of the control value. The presence of a less fluorescent glucuronide conjugate of carboxyfluorescein was not detected in plasma, urine or feces. Fluorescence intensity in the ocular tissues measured, including choriocapillaris, pigment epithelium, retina, ciliary epithelium, iris, and cornea, was not higher for diabetic than for control rats. In addition, there was no indication of localized dye leakage into retina through defects in the pigment epithelial and vascular endothelial barriers or of increased dye entry at the optic disc, a site of blood-retinal barrier discontinuity. Normalization of tissue fluorescence intensity measurements at the different time intervals to compensate for disparity in concurrent plasma dye concentrations resulted in significantly higher levels in diabetic retinas at 1 hr. However, because this difference was not manifest when the plasma dye concentration vs. time integral was used to normalize the data, it is concluded that no greater accumulation of carboxyfluorescein occurs in the retina of diabetic rats over the time period studied.

The uptake of fluorescein-conjugated dextran 70,000 by the small intestinal epithelium of the young rat and pig in relation to macromolecular transmission into the blood.

The macromolecular transmission from the intestinal lumen into the circulation, and its cessation (intestinal closure), were investigated in young rats and pigs in relation to the enterocytes ability to internalize macromolecules. After gavage feeding of FITC-labelled dextran 70,000 (FITC-dextran) and bovine serum albumin (BSA), the uptake of FITC-dextran into the enterocytes was examined by fluorescence microscopy, and the intestinal transmission of both markers was estimated from their blood serum concentrations. In both preclosure rats (14-days old) and piglets (newborn, unsuckled), high serum concentrations of the markers were correlated with the presence of highly fluorescent enterocytes. Although the transmission of the markers to the blood had ceased in postclosure suckling pigs (24-h old), the enterocytes showed a high fluorescence, indicating that the cellular uptake of FITC-dextran was still high. In the 6-days old piglets, only the distal part of the intestine showed uptake of FITC-dextran. In postclosure rats (21- and 30-days old) and in pigs 4-8 weeks old, no fluorescence in the enterocytes and only trace amounts of markers in the serum could be detected. These results reflect differences in the closure process between the species. In the rat, closure is likely to be due to a decrease in the endocytotic activity of the enterocytes, whereas closure in the pig is related to a cessation of the passage of internalized material into the blood (transcytosis).

Effect of chronic partial pancreatic duct obstruction on pancreatic duct secretory pressure and permeability in cats.

Pancreatic secretion during pancreatic duct obstruction results in increased duct pressure. The normally impermeable pancreatic duct becomes permeable to macromolecules the size of pancreatic enzymes after secretion against obstruction. Permeability and morphologic changes may be related to increased secretory pressure during obstruction. We obstructed the main pancreatic duct of cats by 25-100% of its luminal diameter in different groups for 2, 7, or 28 days. Permeability to macromolecules of fluoresceinated dextran (FD) was greatest in cats with less than 75% obstruction compared with cats with greater than 75% obstruction regardless of the duration of obstruction. The frequency of permeability to FD decreased significantly as both the degree and duration of obstruction increased. Secretory pressure also changed according to degree and duration of obstruction. The highest pressures were in cats with complete obstruction at 2 days. Pressure decreased as the degree and duration of obstruction increased. Histologic changes such as acinar lobular atrophy and interstitial fibrosis were most severe in cats with the greatest degree and duration of obstruction. Pressure and permeability changes indicate a greater sensitivity to increased duct pressure than previously thought. These observations may clarify the role of pancreatic duct obstruction in pancreatic disease.

Carboxyfluorescein transfer across the blood-retinal barrier evaluated by quantitative fluorescence microscopy: comparison with fluorescein.

Carboxyfluorescein levels in ocular tissues of normal rats were measured using quantitative fluorescence microscopy and compared with fluorescein levels to determine the extent to which blood-retinal barrier permeability is affected by the difference in lipid solubility of these two dyes. Retinal fluorescence intensity measurements at 2 min after i.v. dye injection were very much lower for carboxyfluorescein than for fluorescein despite similar plasma free dye concentrations. Marked leakage of dye from the optic disc into peripapillary retina was identified. At 1- and 2 hr, retinal levels of the two dyes became more similar, because fluorescein was removed from retina faster than carboxyfluorescein. After sodium-iodate-induced damage of the pigment epithelium, high levels of both dyes were evident in retina, but carboxyfluorescein was localized chiefly within extracellular space whereas fluorescein also densely stained cell somata. The fluorescence intensity levels recorded, which are proportional to the total mass of dye in the tissue, were correspondingly lower for carboxyfluorescein than for fluorescein, indicating that they were markedly affected by the different distribution of the two dyes. To relate tissue fluorescence intensity directly to dye concentration in the extracellular fluid, measurements were obtained from isolated retinas incubated in dye solutions of known concentration. Log-log plots demonstrated a linear relation between fluorescence intensity and medium concentration for both dyes, but retinal fluorescence of carboxyfluorescein, in correspondence with its limited distribution in the tissue space, was consistently less than that of fluorescein. The ratio of carboxyfluorescein to fluorescein fluorescence varied with the retinal layers but was constant for each layer over the concentration range tested. These fluorescence intensity ratios then were used to adjust the in vivo data so that comparison between the two dyes more closely reflected their extracellular dye concentration. With this correction the amount of carboxyfluorescein present in outer retina shortly after dye injection was approx. 1/10 that of fluorescein, indicating that carboxyfluorescein penetrates the pigment epithelium less readily than fluorescein, as expected from the difference in lipid solubility of the two dyes. However, fluorescence of both dyes in retina and presumably in vitreous humor eventually reached similar levels. This is attributed to entry of the dyes at sites of barrier discontinuity, as at the optic disc, and by a difference in their rates of removal from the intraocular compartment.

[Angiography by the oral route using enteric-coated capsules]. / Angiographie par voie orale avec des capsules entériques.

We have performed Retinal Fluorescein Angiograms in three groups of patients who had been previously administered, respectively: intravenous sodium fluorescein, hard gelatin-coated fluorescein oral capsules, and enteric-coated fluorescein capsules. In all groups, we carried out a curve of the dye plasma levels. We concluded that the enteric-coated fluorescein capsules provide effective dye plasma levels for the performance of the angiogram between 40 and 60 minutes later, thus obtaining great sharpness and quality retinal images, much better than those attained with the hard gelatin-coated fluorescein capsules.

Plasma fluorescein decay determination during fluorophotometry.

Two useful methods for determination of the decay curve of non-protein bound fluorescein (NPBF) in plasma up to 1 hour after intravenous fluorescein injection are described and evaluated. The course of NPBF is approximated in method 1 by a sum of two exponential decay functions and in method 2 by a power of time function. The parameters in these functions are calculated with the use of concentration values measured in two blood samples taken at about 5 min. and 60 min. after injection. Calculations in method 1 include the amount of fluorescein injected. The accuracy of each method was evaluated in 7 volunteers by measuring NPBF concentration in 15-28 blood samples taken after fluorescein injection at intervals of 5 min. or less. The mean relative deviation between calculated and measured concentration values amounted to 9.2% +/- 4.3 SD and 12.7% +/- 4.5 SD for method 1 and 2, respectively. The time integral of NPBF concentration in plasma up to one hour after injection was calculated according to the results of both methods and compared with integral values obtained by linear interpolation between concentration values measured in the 15-28 plasma samples. The mean relative deviation for the 7 volunteers amounted at 15 min. to 2.8% and 17% and at 60 min. to 11% and 18% for method 1 and 2, respectively. The maximal difference between the blood-retinal barrier permeability value for NPBF calculated with and without taking glucuronation into account was estimated to be 20% for an average glucuronation percentage of 70% or less.

Role of fluorescein glucuronide and its metabolism in vitreous fluorophotometry.

Rabbits were given fluorescein or fluorescein glucuronide intravenously. Fluorescein and fluorescein glucuronide concentrations in plasma and vitreous samples were measured by high-performance liquid chromatography. Vitreous fluorophotometry was performed using the Fluorotron Master to compare scans after administration of fluorescein and fluorescein glucuronide, and for comparison of in vivo fluorescence with in vitro high-performance liquid chromatography analysis. Fluorescein glucuronide was shown to enter the vitreous as early as 1 hr after injection. Fluorescein glucuronide was the dominant molecule in both vitreous and plasma of all rabbits at 6 hr. Because fluorescein glucuronide has a lower fluorescence than fluorescein, the fluorophotometer overestimates the vitreous concentration of fluorescein after its administration. Since fluorescein is metabolized rapidly to fluorescein glucuronide in man, entry of fluorescein glucuronide into the eye should be considered in measurements of blood-ocular barrier permeability by vitreous fluorophotometry.

Fluorescein and fluorescein glucuronide in the vitreous body of diabetic patients.

Fluorescein (F) and fluorescein glucuronide (FG) were determined in the vitreous of four diabetic patients by a double-filter slit-lamp fluorophotometric technique. Determinations were performed 60-80 min after i.v. injection of fluorescein. F and FG were also determined in plasma ultrafiltrate 5, 15, 30, 60 and 120 min after injection by high-pressure liquid chromatography. The concentration of FG in the vitreous was 3 times that of F. After correction for plasma concentrations of FG higher than those of F, the penetration index of FG through the blood-retinal barrier was found to be twice the penetration index of F. This is not what would be expected if passive transport alone were involved. Accordingly, it is suggested that active transport mechanisms contribute to the movement of F and FG across the blood-retinal barrier.

Fluorescein dilaurate serum test: a rapid tubeless pancreatic function test.

The value of a modified fluorescein dilaurate (FDL) serum test for the detection of pancreatic exocrine insufficiency was investigated in 89 patients with and without pancreatic disease. This test modification with fluorescein serum determination following metoclopramide (10 mg) and secretin (1 U/kg) i.v. injection appeared efficacious in a pilot study in six healthy volunteers. Individual peak fluorescein serum concentration was achieved within 180 min after the test meal in 96% of all subjects studied. Peak fluorescein serum concentration within this time period allowed the best discrimination between normal and abnormal pancreatic function. Sensitivity in detection of chronic pancreatitis was 86% (38 of 44 patients) when the lower normal fluorescein serum concentration was considered 4.5 micrograms/ml (this value corresponds to mean - 2 SD). The specificity of this test in detecting chronic pancreatitis was 100% when healthy controls were considered, but fell to 78% when patients with different gastrointestinal disorders, including those with secondary pancreatic insufficiency, were included. The correlation between serum and urinary fluorescein determination was significant (r = 0.61; p less than 0.01). Duodenal bicarbonate output/h after secretin also showed a significant correlation with peak fluorescein serum concentration (r = 0.79; p less than 0.001).

Permeability of the blood-retinal barrier to carboxyfluorescein in eyes with rhegmatogenous retinal detachment.

Outward and inward permeability of carboxyfluorescein across the blood-retinal barrier were measured fluorophotometrically in seven cynomolgus monkey eyes with experimental rhegmatogenous retinal detachment. Probenecid was used to inhibit outward transport of carboxyfluorescein. The outward permeability was 1.98 +/- 0.31 microliter/min in eyes with retinal detachment and 0.84 +/- 0.15 microliter/min in control eyes with vitrectomy alone (P less than 0.01). The inward permeability, determined separately following intravenous injection, was significantly lower than the outward permeability: 0.14 +/- 0.02 microliter/min for eyes with retinal detachment and 0.04 +/- 0.01 microliter/min for control eyes. Since the outward permeability minus the inward permeability in the presence of probenecid represents that fraction of tracer moving due to fluid flow, it may be concluded that outward flow of fluid across the blood-retinal barrier is a substantial contributor to carboxyfluorescein loss from the vitreous cavity following intravitreal injection.

Fluorescein and fluorescein glucuronide pharmacokinetics after intravenous injection.

The permeability of the blood-retinal and blood-aqueous barriers to fluorescein (F) and the rate of aqueous flow can be estimated by measurements of F in the vitreous, aqueous, and plasma after systemic administration. F is commonly measured by fluorescence, but fluorescein glucuronide (FG), a metabolite of F, also fluoresces. To assess the influence of FG on the quantitation of F by fluorescence, we studied the pharmacokinetics of F and FG for 38 hr in the plasma of five normal subjects given 14 mg/kg of sodium fluorescein intravenously. The plasma and the plasma ultrafiltrate were measured by fluorescence and by high performance liquid chromatography. In our fluorophotometer, FG was 0.124 times as fluorescent as F. F was rapidly converted to FG, and within 10 min the concentration of unbound FG exceeded that of unbound F. The terminal half-lives of F and FG in the plasma ultrafiltrate were 23.5 and 264 min, respectively, so that FG contributed almost all of the plasma fluorescence after 4-5 hr. Because FG was less bound in the plasma than F, the ratio of the fluorescence of the plasma ultrafiltrate to that of the plasma increased with time. The greatest proportion of the total F available to penetrate into the ocular compartments occurred shortly after injection. We concluded that FG is an important contributor to the fluorescence of the plasma ultrafiltrate after intravenous injection and that accurate quantitation of physiologic parameters calculated from the plasma F requires taking this factor into account.

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.

Effect of sorbinil on blood-retinal barrier in early diabetic retinopathy.

To study the effect of sorbinil on the alteration of the blood-retinal barrier, 32 adult-onset, non-insulin-dependent diabetic patients with minimal or no retinopathy were randomly assigned to receive either oral sorbinil (250 mg once a day) or a placebo for 6 mo. All patients underwent fundus photography, fluorescein angiography, and vitreous fluorophotometry before treatment and at 3 and 6 mo after treatment. Vitreous fluorophotometry data showed that the alteration of the blood-retinal barrier increased significantly less in the sorbinil-treated group compared with the placebo group during the 6-mo study period. Side effects were limited to hypersensitivity reactions, with skin rash and fever, in only 2 of the 16 patients who received the drug. These hypersensitivity reactions disappeared with discontinuation of the medication. Aldose-reductase inhibition may play an important role in stabilization of the blood-retinal barrier in early diabetic retinopathy.

Spectral shift of fluorescein and carboxyfluorescein in the anterior chamber of the rabbit eye following systemic administration.

Systemically administered fluorescein is converted to fluorescein glucuronide. The conjugate is fluorescent and interferes with the measurement of fluorescein in the anterior chamber. Carboxyfluorescein is a hydrophilic derivative of fluorescein. In the rabbit, carboxyfluorescein is not converted as readily to a fluorescent metabolite. Thus, carboxyfluorescein has potential advantages as a quantitative fluorophore for studies of aqueous humor dynamics.

Ocular fluorophotometry in the normal- and diabetic monkey.

Normal- and diabetic rhesus monkeys without retinopathy demonstrable by ophthalmoscopy or fluorescein angiography were examined with ocular fluorophotometry to detect alterations in their blood-ocular barriers. All vitreous fluorophotometry values were corrected for fluorescence attributable to background levels and then normalized to a blood fluorescein level of 10 micrograms ml-1. Reproducibility studies demonstrated an average coefficient of variation of 0.17 for all animals combined. Insulin-dependent monkeys, both pancreatectomized and streptozotocin-treated, demonstrated significantly higher posterior vitreous fluorescence levels than either control animals or monkeys treated with streptozotocin that were not insulin-dependent. These results cannot be attributed to differences in fluorescein binding or to vitreous abnormalities. However, 14 out of 24 (58%) of the insulin-dependent animals exhibited posterior vitreous fluorescence values within two standard deviations of the control mean. No correlation was apparent between the vitreous values and age or duration of treatment. No difference in anterior chamber concentrations was found between groups after correction. Our results indicate that alterations in blood-retinal barrier can occur in insulin-dependent diabetic monkeys before development of retinopathy.

Pulmonary absorption of carboxyfluorescein in the rat.

The pulmonary absorption of the fluorescent marker 6-carboxyfluorescein (CF) has been characterized. CF was administered intratracheally (i.t.) as a fluid instillate to pentobarbitone-anaesthetized rats at doses of 0.5 and 2 mg kg-1. The absorption was characterized by both model-independent and model-dependent pharmacokinetic analyses of blood concentration data with reference to previous intravenous (i.v.) studies. The mean fraction available (F) of CF was 90 and 112% with a mean absorption time of 107 and 109 min for the lower and higher doses, respectively. The terminal half-life for the i.t. administered CF (73 and 83 min for the 0.5 and 2 mg kg-1 doses, respectively) was significantly longer (P less than 0.001) than after i.v. dosing (18 min). This indicates a slow pulmonary absorption of CF. Blood concentration-time profiles could not be adequately described by models involving a simple first-order absorption process; a model incorporating two simultaneous first-order inputs gave a much better description, its absorption rate constants differing by almost two orders of magnitude.

Enhancing effect of absorption promoters on percutaneous absorption of a model dye (6-carboxyfluorescein) as a poorly absorbable drug. III. Histological study after addition of various absorption promoters in rats.

We have studied the effect of various absorption promoters on the percutaneous absorption dynamics of 6-carboxyfluorescein (CF) which is a poorly absorbable drug. The absorption of CF was determined by fluorescence photographic image since CF emits a strong light yellow fluorescence. The co-administration of sodium dodecylsulfate (SDS) and 2-mercaptoethanol (MER), the pretreatment with calcium thioglycolate and the co-administration of Azone (AZ) and surfactant (HCO-60) were used to promote the absorption of CF. We have also studied the injury of skin tissue by the absorption promoters. The fluorescence photographic image of rat skin, after the co-administration of SDS and MER and after the co-administration of AZ and HCO-60 were similar to the image with the stratum corneum removed from the skin. These absorption promoters did not affect the histological nature of the rat skin tissue and the recovery experiment showed no injurious effect by these absorption promoters to the skin tissue.