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Concentration-time profiles of fluorescein isothiocyanate (FITC)-conjugated bovine serum albumin and a graded series of FITC-dextrans of 20,000-70,000 molecular weight were measured within the erythrocyte-free plasma layer in individual vessels and at various positions within the interstitial tissue space of mature granulation tissue grown in a rabbit ear chamber. Sodium fluorescein was used as a representative small molecule. The plasma pharmacokinetic data were found to follow a biexponential decay in time. A one-dimensional model of diffusion adequately described interstitial transport. Interstitial diffusion coefficients decreased progressively with Stokes-Einstein radius with values for albumin being significantly reduced from that for a dextran of equivalent hydrodynamic radius. Interstitial diffusion of sodium fluorescein and albumin agreed with a fiber-matrix model, whereas the interstitial diffusion of dextrans more closely corresponded to a pore model.

Hog mucosal heparin (N-sulfate, 0.84 mol; O-sulfate, 1.55 mol; N-acetyl, 0.12 mol; anticoagulant activity assayed by the method of U.S. Pharmacopeia, 161 USP units/mg) or its partially N-desulfated heparin (N-sulfate, 0.71 mol; O-sulfate, 1.47 mol; N-acetyl, 0.12 mol; anticoagulant activity, 117 USP units/mg/ was reacted with 5-isothiocyanatofluorescein in 0.5 M carbonate buffer (pH 8.5) at 35 degrees C for 6 h to yield the corresponding N-fluoresceinylthiocarbamoyl heparins (lambdaem 516 nm, lambdaex 491 nm; degree of substitution 0.006 and 0.013, respectively, anticoagulant activity, 174 and 140 USP units/mg, respectively). The fluorescent heparin (degree of substitution, 0.006; 174 USP units/mg) was injected into rabbits intravenously. The half-life of the fluorescent heparin determined by fluorometry was 24 min, that determined by the clotting time assay was 39 min. The time-course of concentration and the half-life of the fluorescent heparin and of the starting heparin obtained by the clotting time assay were virtually identical.

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.

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.

Stimulated human polymorphonuclear leukocytes (PMNL) have a marked increase in oxidative metabolism, producing reduced oxygen species (e.g., H2O2) that mediate bacterial killing. Previously, quantitation of metabolic responses of PMNL from patients with acute infections employed assays that measure mean activity of the entire PMNL population; such studies reported a modest and highly variable increase in oxidative metabolic responses of such "toxic" PMNL compared with normal cells. To assess metabolic capability of PMNL from 51 patients with acute bacterial infection, we employed a quantitative flow cytometric assay of H2O2-dependent oxidative product formation, the intracellular oxidation of 2',7'-dichlorofluorescin (DCFH). After stimulation by phorbol myristate acetate, the PMNL of patients demonstrated an increase in mean DCFH oxidation (315 +/- 14 and 180 +/- 4.5 amol/cell, patients and controls). Hexose monophosphate shunt activation was similarly increased in stimulated PMNL from bacteremic patients. These data are comparable with previous studies of mean metabolic activities of toxic PMNL. However, these mean values underestimate the quantitative responses of the hyperresponsive ("primed") PMNL within a mixture of normal and primed PMNL in the patients' blood. The flow cytometric assay demonstrated that the PMNL of the patients were composed of two populations. One population of PMNL had normal oxidative responses; the other "primed" population had up to 4.6 times the oxidative product formation of normal cells. Similar priming of circulating PMNL was caused by infection with gram-positive or gram-negative staining bacteria or by Candida species. The proportion and oxidative ability of the primed PMNL occurred independently of the number of juvenile neutrophil forms and independently of "toxic" morphologic changes of Wright's-stained PMNL. On the average, 40% of the PMNL of patients were primed, but the size of the primed PMNL population varied widely between patients (range 0 to 80%). This variable subpopulation may explain the variability of mean responsiveness of the PMNL of patients reported previously. Moreover, the marked increase in oxidative metabolic capability of the primed PMNL may be a significant component of the host response to acute infection. It could also contribute to the damage to host tissues such as pulmonary vascular endothelium during bacteremia.

Fluorophotometry was used to study the permeability of the blood-retinal barrier in six monkeys with stable rhegmatogenous retinal detachments. Fluorescein transport was inhibited by probenecid. The rate of fluorescein disappearance (KvVv) was determined following intravitreal injection. In a separate experiment the equilibrium vitreous-plasma concentration ratio (Cv/Cp) was determined following intraperitoneal administration. Expressed in equivalent volumes of vitreous, the rate of fluorescein diffusion across the blood-retinal barrier (K'vVvCv/Cp) was 0.29 microL/min in control eyes and 0.73 microL/min in detached eyes. The rate of fluid movement across the blood-retinal barrier (K'vVv) [1 - (Cv/Cp)] was 2.89 microL/min in control eyes and 6.38 microL/min in detached eyes. Posterior movement of fluid contributes to retinal apposition under normal conditions and accounts for the rapid resolution of retinal detachment following closure of the retinal hole.

Hypochlorous acid (HOC1) rapidly chlorinates fluorescein compounds forming, sequentially, the corresponding 4'-chlorofluorescein and 4',5'-dichlorofluoresceins. Chlorination by cell-free myeloperoxidase-catalyzed chloride peroxidation systems gives rise to these compounds as well as variable amounts of isomeric compounds chlorinated in the 2'- and 2',7'-positions. The fluorescence intensity of the dianionic form of the dye is partially quenched upon chlorination, and its proton equilibrium constants are shifted to more acidic values. Fluorescein covalently bound to zymosan (5-isothiocyanatofluorescein-zymosan) also formed these products when the unopsonized particles were incubated with phorbol myristate acetate- or N-formyl-methionyl-leucyl-phenylalanine-stimulated human neutrophils. This reaction was associated with a fall in fluorescence intensity, which was not observed when cells from individuals with chronic granulomatous disease or myeloperoxidase deficiency were used or when azide or catalase were added to the reaction medium. Fluorescent changes accompanying phagocytosis of serum-opsonized 5-isothiocyanatofluorescein-zymosan were also consistent with chlorination of the label; the changes were shown to be myeloperoxidase-dependent by use of myeloperoxidase-deficient or azide-treated cells. Oxidative bleaching of the structurally similar sulfonphthalein dyes by HOCl also occurs at rates which parallel the dye basicities. Results are discussed in relation to the use of fluoresceinated particles and sulfonphthalein dyes in the measurement of intraphagosomal acidification.

The percutaneous absorption of drugs was investigated in rats by measuring plasma levels, using mainly 6-carboxyfluorescein (CF) as a model of poorly absorbable drugs. Azone (AZ), a new useful promoter for the percutaneous absorption of drugs, was used instead of dimethylsulfoxide. We have examined the effects of the solubilized state and concentration of AZ on the percutaneous absorption of CF. AZ was dissolved with the aid of surface-active agents, beta-cyclodextrin (CD) or dimethyl-beta-cyclodextrin (DMCD). When AZ (2 v/v%) was dissolved completely by a surface-active agent (HCO-60: polyoxyethylene hardened castor oil derivative), plasma CF levels showed the highest value. Plasma CF levels following the administration of CF with AZ which formed a complex with CD or DMCD were scarcely increased as compared to that of CF alone. In the case of fluorescin (FL), which has a higher partition coefficient than CF, the percutaneous absorption of FL was more enhanced by the addition of AZ than in the case of CF.

The binding of fluorescein and 5- and 6-carboxyfluorescein by human serum proteins was measured at 37 and 4 degrees C by equilibrium dialysis. The equilibrium association constants (KA) for fluorescein were 3.7 X 10(3) and 7.1 X 10(3)M-1, and for carboxyfluorescein were 3.5 X 10(3) and 7.5 X 10(3)M-1 at 37 and 4 degrees C equilibrium dialysis data, was 3.9 X 10(3) binding sites in human serum, determined from the 37 degrees C equilibrium dialysis data, was 3.9 X 10(-3)M for fluorescein and 3.3 X 10(-3)M for carboxyfluorescein. Utilizing these binding parameters it was calculated that a maximum of 93.5% of the total fluorescein and 92.0% of the carboxyfluorescein would be bound by undiluted human serum proteins at 37 degrees C. Experimental binding data obtained after prolonged equilibrium dialysis (four days) at low total fluorochrome concentrations (1.5 X 10(-4)M or less) indicated that 93.1 +/- 1.0 (S.D.)% of the fluorescein and 90.1 +/- 0.7% of the carboxyfluorescein were bound at 37 degrees C by undiluted human serum proteins. Stopped-flow kinetic spectrophotometric studies of the changes in absorptivity at 487-488 and 510 nm that occurred when the fluorochromes were bound by human serum proteins, indicated that the fluorescein and carboxyfluorescein binding reactions were 99% complete within 0.65 and 1.72 sec. These had second-order association rate constants at 25 degrees C of 3.0 X 10(3) and 1.5 X 10(3)M-1 sec-1, respectively. These findings offer a basis for calculation of bound and free fluorescein and carboxyfluorescein in vivo in human subjects.

We compared insulin-dependent diabetic patients with minimal (16 eyes of 9 patients) or no retinopathy (45 eyes of 27 patients) to normal volunteers (20 eyes of 12 subjects) using a commercial vitreous fluorophotometer and different procedures for artifact correction. The influence of background autofluorescence was minimized through the use of a software program that subtracted a fluorophotometric scan obtained before administration of fluorescein from that obtained after its injection. We also compared two programs designed to minimize the contribution of the chorioretinal peak spread function to the readings in the vitreous. The fluorescein concentration in the posterior vitreous was then averaged within two different regions. We then assessed the influence of these data-processing methods on the spread of the results of the different groups. The clinical study showed that only the posterior vitreous concentration of fluorescein is relevant in the evaluation of the blood-retinal barrier. However, since there is a gradient of fluorescein concentration in the posterior vitreous, one needs a scanning device so that one can measure at a precise location in front of the retina. The posterior vitreous concentration of fluorescein was significantly increased in diabetic subjects with one or no aneurysms as compared with normals. Moreover, the eyes with minimal retinopathy, as judged by the presence of microaneurysms, had higher values than those without retinopathy. The clear differences among these three groups were not present when the midvitreous values were used.(ABSTRACT TRUNCATED AT 250 WORDS)

A modified slit lamp fluorophotometer was used to determine fluorescein concentration changes in the perilymph, cerebrospinal fluid and blood of chinchillas after intravenous injection of 0.2 ml of fluorescein sodium. This new technique provides a means of determining quantitative changes of fluorescein concentration in the perilymph without the need to withdraw fluid samples through the round window membrane of cochlear wall. Fluorescein was observed to enter the perilymph between 1 and 2 minutes after injection, and it reached its peak concentration in the mean time of 23 minutes. The mean peak concentration was 4.20 X 10-6 g/ml. Both increasing and decreasing fluorescein concentration changes in the perilymph followed an exponential time course. Although the observations of cerebrospinal fluid fluorescence were thought to represent a composite of the fluorescence of the cerebrospinal fluid itself and the underlying brain stem blood vessels, the peak fluorescence did not exceed that observed in the perilymph. These observations support the view that most of the perilymph is produced in the cochlea by ultrafiltration from the cochlear blood vessels. The slit lamp fluorophotometer appears to be a satisfactory means of recording fluorescein concentration changes in the perilymph without disturbing the cochlear physiology by penetrating the labyrinth to obtain fluid samples.

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.

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 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.

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.

A method to determine fluorescein in human plasma is described. By aid of ultrafiltration a separation between fluorescein bound to plasma proteins and fluorescein free in the water is obtained. Both fractions are quantitated. Fluorescein is bound to plasma proteins. The protein binding is reversible, not sensitive to practically appearing changes in pH, temperature and gas tensions. For a normal person it appeared that at a total plasma concentration between (10(-6) - 10(-4))g . ml-1 approximately 15% was free while at a total concentration of 10(-3) g . ml-1 45% was free, pointing towards a limited amount of protein binding sites.

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.

In connection with corpus vitreum fluorophotometric examinations fluorescein was injected iv and fluorescein in the blood stream was studied with respect to protein binding and elimination kinetics. It was shown by ultrafiltration that in blood obtained during the examination most of the fluorescein was protein bound, and only a minor fraction (10-20%) was free in the water phase. While the free fraction was approximately constant over a wide concentration range when studied in vitro the free fraction increased by a factor of about 2 during a 2 h examination period in vivo. The curve describing the elimination of fluorescein from blood showed a polyexponential decline course. The area under this curve showed only a small variation for the same normal person examined 3 times over a period of 6 months. The area under the bolus has a magnitude, which is 30-40% of that under the rest of the curve.

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.

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.

A variety of mechanical (intensity of the light source, the width and the angle of the exciting source) and physiological factors (retinal pigmentation, vitreous fluorescence) and many systemic factors affect the results of vitreous fluorophotometry. These factors are discussed with respect to identifying those that are inconsequential and those that are significant. Methods to compensate for the important factors are described.

We have investigated the promotive effect of various agents on percutaneous absorption of 6-carboxyfluorescein (CF), a water-soluble fluorescent dye, as poorly absorbable drugs. The absorption of CF was determined by measuring rat plasma CF levels. As an absorption promoter, several reagents such as surface-active agents, protein solubilizers and permeation promoters were used. The used concentration of the reagents was determined so as not to make a trauma on the skin. As results, plasma CF levels following the co-administration of 0.05 w/v% sodium dodecyl sulfate and 0.1 v/v% 2-mercaptoethanol showed the highest values. Plasma CF level was increased 40 times as compared to that of control experiment and was increased 9 times as compared to that of pretreatment with 4 w/v% calcium thioglycolate which was reported previously as a strong absorption promoter for theophylline by us. When the stratum corneum, having a barrier function for percutaneous absorption of many compounds, was removed mechanically, plasma CF levels of control experiment and pretreatment with 4 w/v% calcium thioglycolate were increased remarkably. However, plasma CF level after the co-administration of 0.05 w/v% sodium dodecyl sulfate and 0.1 v/v% 2-mercaptoethanol did not show a considerable difference as compared to that of the case with the presence of the stratum corneum.

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.

Forty-six subjects (20 chronic pancreatitis, 7 chronic liver disease, 7 recovered from acute pancreatitis, 2 Crohn's disease, and 10 healthy controls) classified by S-C test as having normal pancreatic function (26 subjects), or moderate (10 subjects) and severe (10 cases) pancreatic insufficiency, were given, on different days, 1 g of oral PABA or 348 mg of oral fluorescein dilaurate. At the 1st, 2nd, and 4th hours (PABA) and the 2nd, 4th, and 6th hours (fluorescein) serum samples were taken for assay. In the presence of severe exocrine pancreatic insufficiency, the sensitivity of the fluorescein serum levels was higher than that observed for the PABA (100% and 80%, respectively), and quite similar to that shown by the urinary tests (100% and 70%, respectively). On the contrary, in presence of moderate pancreatic insufficiency, both the urinary test (pancreolauryl and (PABA) give a sensitivity higher than that found in the serum tests (30-40% and 10-30%, respectively). The parallel combination of both the serum or urinary tests does not significantly improve the sensitivity of the single test. These results suggest that the serum PABA and serum fluorescein tests can be valid choice when a prolonged urinary collection is difficult, i.e., in children and in elderly patients. However, the slight diagnostic gain does not justify the routine use of both urinary and serum tests.

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.

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.

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).

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.

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).

Since serum fluorescein levels are altered in various experimental states, we examined the relationship between serum and vitreous fluorescein levels. We also injected streptozocin, alloxan, and alloxan with glucose pretreatment do determine whether alterations in the blood-retinal barrier are directly attributable to drug toxicity. When corrected for serum levels, one- and two-hour vitreous fluorescein levels increased above prediabetic values two days after streptozocin or alloxan administration; two-hour readings were higher. Rats treated with alloxan plus glucose did not become diabetic or show elevated vitreous fluorescein levels. Insulin treatment of alloxan-induced diabetic rats resulted in normal vitreous readings without normoglycemia. These results suggest that vitreous readings should be corrected for serum levels and that observations at two hours could be more sensitive than at one hour. Furthermore, the observed alterations in blood-retinal barrier function are not attributed to drug toxicity.

The blood-retinal barrier plays an important part in the processes of retinal pathophysiology. A new clinical method for the study of the blood-retinal barrier by vitreous fluorophotometry appears to satisfy the necessary requirements in that it is quantitative and shows good reproducibility and high sensitivity. The application of this method to a series of diabetic patients with apparently "normal" fundi revealed the presence of a significant breakdown of the blood-retinal barrier in the early stages of retinal involvement in diabetes. The extent of the breakdown can be measured allowing for comparative and evolutionary evaluations. The disturbance of the blood-retinal barrier, as evidence by vitreous fluorophotometry, appears before microaneurysms or capillary closure can be demonstrated by fluorescein angiography.

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 binding of 5 drugs to plasma proteins from patients with alcoholic liver disease has been studied by in vitro techniques and compared to that from normal subjects. The binding of quinidine is decreased in such patients. For this reason, if in patients with alcoholic liver disease treated for coincidental arrhythmias with quinidine, plasma quinidine concentrations are measured, allowance for the decreased binding must be made in interpreting the concentration measurements. Diphenylhydantoin binding was normal or near normal in most of the patients' plasma samples. The other drugs tested, fluorescein, dapsone, and triamterene, had decreased binding in the patients' plasma samples.

The exchange of fluorescein isothiocyanate-labeled dextrans ranging in weight-averaged molecular weight from 19,400 to 160,000 and 125I-bovine serum albumin (BSA) between dialysis fluid (5% BSA in Krebs-Ringer solution) in the peritoneal cavity and the plasma was studied in anesthetized female Sprague-Dawley rats. Plasma and peritoneal samples were collected for 3-4 h after either 1) an intraperitoneal injection of dialysis fluid with tracer or 2) an intravenous injection of tracer material simultaneously with an intraperitoneal injection of dialysis solution without tracer. Analysis of the data by means of a mathematical model of the transport process suggests a functional asymmetry in transport of large molecules across the blood capillary wall. Substances injected intravenously have a net transport from the blood capillaries to the peritoneal cavity. Substances of molecular weight greater than or equal to 39,000 transport from the cavity to the plasma via peritoneal lymphatics; 19,400 molecular-weight dextran transports from the cavity to the plasma primarily via lymphatics with some blood capillary uptake. Tissue diffusivities and capillary mass transport coefficients are derived for the substances tested.

In an attempt to enhance delivery of liposome contents into cells, we tested the effect of lysophosphatidylcholine on transfer of the fluorescent dye, carboxyfluorescein, from small unilamellar and large multilamellar vesicles to human lymphocytes. Dioleoyl phosphatidylcholine and dioleoyl phosphatidylcholine-lysophosphatidylcholine small unilamellar vesicles with varying lipid ratios were prepared and characterized. In the presence of lysophosphatidylcholine, small unilamellar vesicles were slightly smaller and more leaky than those made without lysophosphatidylcholine. Lysophosphatidylcholine induced less leakage in large multilamellar vesicles. It did not show any appreciable effect on transfer of liposome contents, whether included as part of the liposomal bilayer (of unilamellar or multilamellar vesicles) or added exogenously together with small unilamellar dioleoyl phosphatidylcholine vesicles.

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.

The in vitro plasma protein binding was determined in nine maintenance hemodialysis patients who later underwent renal transplantation. The organic acid fluorescein (10 micrograms/ml) or the organic base quinidine (5 micrograms/ml) was added to the pre and post transplant serum of these patients. Drug concentrations were measured spectrophotofluorometrically after equilibrium dialysis. The results were compared with the plasma protein binding of eight normal volunteers. The patients on maintenance hemodialysis had lower plasma protein binding of fluorescein than normals (78 +/- 5% vs 89 +/- 4, p less than 0.001). Plasma protein binding improved significantly after renal transplantation (85 +/- 3, p less than 0.01) but was still lower than in normals (p 0.05). Plasma protein binding of quinidine was not significantly different than in normal volunteers (77 +/- 8%) either prior to (72 +/- 10%) or after (73 +/- 12%) kidney transplantation. Plasma protein binding of quinidine remains unaffected by renal transplantation. However, the abnormal plasma protein binding or organic acids in chronic renal failure may be significantly improved by renal transplantation.

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.

Some aspects of the pharmacokinetics of fluorescein have been studied under the conditions of the fluorescein dilaurate test (Pancreolauryl-Test) in healthy volunteers. Dependence of fluorescein excretion on urine volume was investigated in a retrospective study in 370 patients. For intravenously administered fluorescein mean Cmax was 10.9 micrograms/ml with a mean elimination half-life of 286 min. For orally administered fluorescein sodium mean Cmax was 3.5 micrograms/ml with a tmax of 120 min and a t 1/2 of 267 min. Bioavailability of fluorescein by oral administration was 99%. By contrast, fluorescein from fluorescein dilaurate showed a 56% bioavailability under the conditions of the test with a Cmax of 1.8 micrograms/ml, a tmax of 270 min and a tt 1/2 of 246 min. Following enteral absorption of fluorescein hepatic extraction and enterohepatic circulation via the bile occurs, but although the concentrations of fluorescein in the bile may exceed those in the urine the absolute amount is likely to be small. In spite of the enterohepatic circulation fluorescein cleared from the urine within 24 h indicating that no delay between Part 1 and Part 2 of the test seems necessary. However, an adequate urine flow must be maintained throughout the test since a renal clearance/urine flow relationship exists, with fluorescein excretion being increased with increasing urine volume.

In contrast to situation found in other cell types, no linear dependence of product fluorescence vs time is observed when fluorescein diacetate (FDA) is hydrolysed by erythrocytes and hemolysates. The rate of hydrolysis is increased by high concentrations of sucrose suggesting a positive effect of viscosity on the rate of the reaction. These peculiarities can be explained by assumption of a two-step hydrolysis of FDA. The FDA-hydrolytic activity decreases with increasing cell density (age).

The penetration of systemically administered fluorescein sodium into the eye depends on several factors. One important variable is the unbound plasma fluorescein concentration. We measured the in vitro plasma binding of fluorescein in 200 persons using fluorescence polarization. We demonstrated considerable variability in the degree of plasma binding and also found significant differences between normal persons and diabetic and nondiabetic patients.