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PubMed1991

Differential spectrofluorometry in the human vitreous: blood-retina barrier permeability to fluorescein and fluorescein glucuronide.

A method is described for the separate quantitation of fluorescein and fluorescein glucuronide in the vitreous by differential spectrofluorometry. An ocular fluorometer was equipped with monochromatic laser excitation at two rapidly interchangeable wavelengths. The data analysis accounts for absorption of light in the cornea, lens, and extrinsic ocular fluorophores. Examination of seven patients with insulin-dependent diabetes and different degrees of diabetic retinopathy demonstrated that both fluorescein and fluorescein glucuronide enter the eye through the blood-retina barrier. The mean ratio between the permeabilities of fluorescein glucuronide and fluorescein was 0.9 (range, 0.3-1.9). Thus, differences in the molecular size and lipid solubility of the two substances appear to be of little or no importance for their inward penetration of the barrier. No association was found between the relative permeability and the degree of retinopathy.

Adult↗
PubMed1990

Intracellular turnover of fluorescein diacetate. Influence of membrane ionic gradients on fluorescein efflux.

The influence of the membrane ionic gradient on the efflux of Fluorescein after intracellular turnover of Fluorescein diacetate was studied in HeLa cells. The kinetics of Fluorescein efflux was monitored by determining with flow cytometry the decrease in fluorescence intensity of single cells. Alterations of the Na+ and K+ gradients were induced experimentally by using ouabain, ionophores or buffers in which the ion concentration was modified. The sodium gradient was also altered by using Na(+)-co-transported amino acids. Independent evidence of these changes was obtained with a potential-sensitive indicator, 3,3'-dihexyloxacarbocyanine iodide. Conditions inducing a reduction or dissipation of the ionic gradients caused a decrease in the rate constant of Fluorescein release. In contrast, enhancement of the gradients increased the efflux rate. These results indicate that the release of Fluorescein from living cells is influenced by the membrane potential. Thus, the turnover of Fluorescein diacetate may provide a useful technique for assessing changes in membrane permeability properties related to ionic gradients.

Biological Transport↗
PubMed1992

Oral fluorescein angiography and fluoroscopy: determination of plasma fluorescein levels and clinical application.

One hundred oral fluorescein angiographic explorations were performed in 85 patients of both sexes, mean age 55.57 years, and range 8 to 77 years. Fluorescein was administered as one 250-mg (15 patients) or 500-mg (70 patients) capsule per 15 kg of body weight. In 15 fasting and 15 postprandial patients administered 500-mg capsules, no significant differences by group were found between fluorescein plasma levels. Fundus fluorescence under slitlamp illumination was poor for patients receiving the lower dosage of fluorescein, whereas at the higher dosage it was visible in all but one patient. The best filters were found to be a Kodak-Wratten 47A for the excitation filter and Kodak-Wratten 8 for the barrier filter. With a conventional fundus camera and fluorescein angiography filters, good photographs were achieved in all cases. The integrity of the blood-retina barriers was explored for various chorioretinal pathologies.

Administration, Oral↗
PubMed1982

Report on the presence of a toxic substance, dimethyl formamide, in sodium fluorescein used for fluorescein angiography.

The revelation that intravenous sodium fluorescein is not all that it might seem to be may be a significant finding in the light of the adverse reactions to fluorescein that have been previously reported. Analysis of commercially prepared intravenous sodium fluorescein by mass spectroscopy and nuclear magnetic resonance has indicated that an industrial solvent used in the manufacturing process has not been eliminated. Dimethyl formamide is an industrial solvent with a maximum acceptable exposure level of 10 parts per million for dermal contact. It has been found in quantities of 5000 parts per million in the fluorescein for intravenous use. This investigation was prompted by a significant increase in the adverse reactions in patients receiving intravenous fluorescein in the retinal photographic unit at the Manchester Royal Eye Hospital.

Dimethylformamide↗
PubMed1985

Drug absorption from inhalation aerosols administered by positive-pressure ventilation. II: Effect of disodium fluorescein aerosol particle size on fluorescein absorption kinetics in the beagle dog respiratory tract.

Solid, polydispersed disodium fluorescein aerosols (MMDae = 1.1, 3.5, and 4.4 micron) were administered under the same respiratory regime, direct to the respiratory tracts of two beagle dogs by positive-pressure ventilation. Subsequent to aerosol administration, plasma fluorescein concentrations were determined after sampling from an indwelling cannula. The amount absorbed as a function of time was estimated from these and additional data collected from intravenous control experiments in the same animals. Fluorescein absorption from the respiratory tract was apparently a first-order process, the rate increasing directly with the bioavailable dose. First-order rate constants differed but appeared unrelated to aerosol particle size, possibly reflecting similarities in their regional deposition in the canine lung. The average value for the absorption half-lives in the dogs were 19.3 and 12.2 min, showing that even lipophobic solutes such as the fluorescein dianion, are absorbed extremely rapidly via the lung. In one dog, the rate constant for fluorescein absorption after intratracheal instillation of a solution of the disodium salt was within the range of those following aerosol administration. Possible explanations are discussed.

Absorption↗
PubMed1999

Fluorescein kinetics in interstitial fluid harvested from diabetic skin during fluorescein angiography: implications for glucose monitoring.

BACKGROUND: Glucose monitoring based on sampling skin interstitial fluid (ISF) is being developed as an alternative to fingerstick blood glucose monitoring. Time delays between rapidly changing levels of glucose in blood and interstitial fluid have been reported in the literature to be between 5 and 20 minutes. This study investigated the time delay between the injection of a small molecular weight fluorescent tracer into the circulation and interstitial fluid. METHODS: Diabetic subjects undergoing fluorescein angiography were studied. Skin ISF was sampled using a proprietary microporation and harvesting process. ISF was drawn through micropores created in the stratum corneum. After intravenous injection of sodium fluorescein, samples of ISF were drawn from 2 sites for 30 seconds over a period of 20 minutes. Fluorescence levels in ISF were measured with a fluorometer and used to create ISF fluorescein concentration versus time profiles. RESULTS: The ISF fluorescein versus time profiles were characterized by a rapid rise followed by a slow decay. The time to peak of the ISF fluorescein concentration ranged from 2-4 minutes for the patients studied. CONCLUSIONS: Intravenous injection of a bolus of low molecular weight fluorescent tracer was used to estimate the time delay between changing glucose levels in blood and ISF. The results indicate that the ISF sampling technology utilized here is capable of tracking rapidly rising levels of blood glucose.

Adult↗
PubMed1997

Effect of consecutively applied fluorescein eye drops on corneal and aqueous concentrations of fluorescein.

The main objective of this study was to determine the intraocular concentration of fluorescein after applying five eye drops with only 1-min intervals in human eyes. The concentration of fluorescein in corea and aqueous was studied with fluorophotometry in 11 healthy volunteers. Five 10-microliter drops of 2% fluorescein solution were administered with 1-min intervals, and the intraocular concentrations that were achieved were compared with those obtained with just one eye drop. Five consecutive fluorescein drops caused a 2.6- to 3.1-fold increase in fluorescein concentrations of the anterior chamber 1-8 h after application compared with a single eye drop (p < 0.05). Corresponding values for the cornea were 2.1-2.8. As a conclusion, administration of five eye drops applied consecutively significantly increases intraocular drug concentration during 8 h in humans.

Adult↗
PubMed1997

Fluorescent dots in fluorescein angiography and fluorescein leukocyte angiography using a scanning laser ophthalmoscope in humans.

PURPOSE: The purpose of the study is to disclose the nature of fluorescent dots and segments traditionally observed with fluorescein angiography (FA) using a scanning laser ophthalmoscope (SLO 101; Rodenstock, München, Germany). The authors developed a new method, called fluorescein leukocyte angiography (FLA), to display directly the movement of leukocytes in human retinal vessels. METHODS: Fluorescein angiography was performed on two normal volunteers using a scanning laser ophthalmoscope and fluorescent dots and segments were observed. Fluorescein leukocyte angiography, using an injection of fluorescent buffy coat layer from which the fluorescent plasma and nonfluorescent erythrocytes have been removed externally, was performed on seven normal volunteers. Injection fluid smears were examined through a fluorescent microscope. Peripheral blood smears taken during midphase of FA and FLA also were examined. In addition, 15 early-phase FAs of central serous chorioretinitis (CSC) were studied retrospectively. RESULTS: In the FAs of normal volunteers, fluorescent dots were detected only in perimacular capillaries at early phase. Eight of the 15 CSC FAs examined showed both fluorescent dots and segments. In the FLAs, fluorescent dots were detected in whole retinal vessels for more than 30 minutes. Fluorescent segments were observed in FA but not in FLA. Injected fluid smears from one FLA showed fluorescent leukocytes and small platelets. However, in peripheral blood smears of the FLA, leukocytes and platelets were more visible and exhibited higher contrast than those of an FA due to background plasma fluorescence. The mean velocity of 21 flowing leukocytes in perifoveal capillaries was 1.37 +/- 0.35 mm/second in 2 FAs and that of 89 flowing leukocytes was 1.41 +/- 0.29 mm/second in 7 FLAs. CONCLUSIONS: The authors' observations suggest that fluorescent dots in scanning laser ophthalmoscope imaging are fluorescein-stained leukocytes, whereas fluorescent segments are the hyperfluorescent plasma that is located between rouleaux formations of erythrocytes. The velocity of the fluorescent dots could be measured in the perimacular capillaries by either FA or FLA; however, only FLA can display the flow of fluorescent leukocytes in large vessels.

Adult↗
PubMed2000

Preparation of Na,K-ATPase specifically modified on the anti-fluorescein antibody-inaccessible site by fluorescein 5'-isothiocyanate.

Specific labeling is required for energy transfer measurements and to avoid artifacts in the use of fluorophores as reporter groups. Therefore, a method for specific modification by one of the most popular reagents for P-type ATPases (fluorescein 5'-isothiocyanate) has been developed. Sulfhydryl reagents protected against modification of cysteine residues, and treatment with dithiothreitol eliminated a slow doubling of the fluorescence of conventionally modified Na,K-ATPase upon dilution that is attributed to disappearance of self-energy transfer. Removal of nonspecifically bound fluorescein was also confirmed by titration of the modified Na, K-ATPase with anti-fluorescein antibody and by time resolution of the fluorescence change when the modified enzyme was mixed with Na(+) in a stopped-flow instrument. The only fluorescence change when specifically modified Na,K-ATPase was mixed with Na(+) was the signal from fluorescein at the antibody-inaccessible, substrate-protectable site that reports the conformational change in unphosphorylated enzyme. The magnitude of the fluorescence change reporting the conformational change increased from between 8 and 12% to between 25 and 30% without affecting the kinetic constants estimated from titrations with Na(+) and K(+). The method should be generally applicable to the preparation of specifically labeled P-type pumps for use in kinetic and equilibrium titrations or energy transfer measurements.

Animals↗
PubMed1976

Fluorescein in the human optic disc. II. The fluorescein appearance rate.

Automatic intravenous infusion of fluorescein with constant speed during 25 sec is followed by a gradual increase of fluorescein in the retinal arteries and in the disc tissue. The slope of this increase Sd divided by the maximum fluorescein Md in an area of the disc Sd/Md is called Fd, the same for a retinal artery is called Fr. Fr was found to be independent of intraocular pressure in the range examined (mean pressures 15--30 mm applanantion). Fd/Fr = omega, the "fluorescein appearance rate" is correlated to the linear velocity of blood in the capillaries of the observed disc area and hence to its circulation. omega was found to be diminished in 6 out of 9 cases with ocular tension between 26 and 38 mm applanation, but not in 3 cases. It was not diminished in 4 cases with 22-25 mm applanation. Further studies are necessary to elucidate these findings suggesting some adaptation of disc circulation to intraocular pressure but not proving it: fluorescence from vessels behind the lamina cribrosa has the tendency to seemingly "normalise" omega.

Adult↗
PubMed1987

The transcapillary exchange of sodium fluorescein in ischaemic limbs measured by fluorescein flowmetry.

The transcapillary exchange of sodium fluorescein in the skin of 36 extremities (19 patients) with arterial occlusive disease was measured using two different ways of analysing the tissue fluorescence, fluorescein flowmetry (FF) and dynamic fluorescein angiography (FA). 7 mg sodium fluorescein/kg body weight was given intravenously. With FF, the transcapillary exchange of sodium fluorescein is expressed in arbitrary units as a fluorescence index. With FA, the time interval from the bolus injection to the first appearance of the fluorescence in the tissue is measured. There was an inverse correlation between pain and fluorescence index. All eight extremities with a fluorescence index of 0.001 density units/s or lower needed amputation, whereas all extremities with higher indices were viable. All extremities requiring amputation had an appearance time of 150 s or more, which contrasted with those remaining viable, having an appearance time of 120 s or less. The results indicate that FF as well as FA are appropriate methods to measure a 'nutritive' transport of solutes in the skin. Theoretically, however, if quantitative values for blood flow are required, we recommend FF, this method being more adequate, since it is influenced both by the fraction of cardiac output distributed to the tissue and by cardiac output itself. If, however, only a prognosis of a limb's viability is needed, FA is sufficient, being easier to perform, only requiring the measurement of the appearance time.

Adult↗
PubMed1986

A new method for using fluorescein to demonstrate oto- and rhinoliquorrhea. I. Sample preparation by electrophoresis and photometric identification of fluorescein.

We have described a new method for the identification of sodium fluorescein in cases of cerebrospinal fluid (CSF) leakage. To take samples, small sponges of Merocel are placed into the nostrils or external auditory canal, as indicated. They are left in situ for 12h after intrathecal injection of 5% sodium fluorescein. Fluorescein is identified by electrophoretic separation on 1% agarose gel and demonstration with the fluorescence photometer. This method of testing has various advantages: the use of small sponges enables collections of minimal amounts of CSF; potential sources of disturbance (e.g. hemoglobin, etc.) are eliminated; false-positive results are avoided; the time needed for sample analysis lasts only 10 min. All necessary materials and equipment, as well as the taking and analysis of samples, are described in detail. The method used was tested both clinically on patients and in the laboratory and its sensitivity is clearly illustrated.

Cerebrospinal Fluid Otorrhea↗
PubMed1987

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.

Blood-Retinal Barrier↗
PubMedDate not supplied

Antibody networks and imaging: elicitation of anti-fluorescein antibodies in response to the metatypic state of fluorescein-specific monoclonal antibodies.

Studies are described regarding generation of anti-hapten antibodies starting with a monoclonal Ig immunogen in the ligand-induced conformation or metatypic state. Liganded monoclonal Ab1 antibodies represent the unique feature of the study since previous reports investigating internal imaging in the original Idiotype Network Hypothesis [Jerne, 1974 (Ann. Immun. 125C, 373-389)] were based on the non-liganded or idiotypic state [as reviewed in: Rodkey, 1980 (Microbiol. Rev. 44, 631-659); Kohler et al., 1979 (In: Methods in Enzymology: Antibodies, Antigens and Molecular Mimicry, pp. 3-35); Greenspan and Bona, 1993 (FASEB J. 7,437-444)]. Affinity-labeled liganded murine monoclonal anti-fluorescein antibodies served as immunogens administered both in the syngenic and xenogenic modes to determine if the metatypic state elicited anti-hapten antibodies through imaging-like mechanisms. Polyclonal and monoclonal anti-Ab1 reagents in various hosts were assayed for anti-fluorescein and/or anti-metatype specificity. Significant anti-fluorescein responses were measured indicating that the metatypic state directly or indirectly stimulates an anti-hapten antibody population.

Affinity Labels↗
PubMed2003

Using bifunctional polymers presenting vancomycin and fluorescein groups to direct anti-fluorescein antibodies to self-assembled monolayers presenting d-alanine-d-alanine groups.

This paper describes the synthesis of bifunctional polyacrylamides containing pendant vancomycin (Van) and fluorescein groups, and the use of these polymers to direct antibodies against fluorescein to self-assembled monolayers (SAMs) presenting d-alanine-d-alanine (dAdA) groups. These polymers bind biospecifically to these SAMs via interactions between the dAdA and Van groups and serve as a molecular bridge between the anti-fluorescein antibodies and the SAM. The binding events were characterized using surface plasmon resonance spectroscopy and fluorescence microscopy. The paper demonstrates that polyvalent, biospecific, noncovalent interactions between a polymer and a surface can be used to tailor the properties of the surface in molecular recognition. It also represents a first step toward the design of polymers that direct arbitrarily chosen antibodies to the surfaces of cells.

Acrylic Resins↗
PubMed1975

Fluorescein angiography of canine conjunctival auto- and allografts. Analysis by sequential, still photographic recording of the spreading of fluorescence over the graft surfaces following intravenously administered fluorescein.

Two autologous and 4 allogeneic conjunctival grafts have been transplanted to the corneal-scleral region in 6 dogs. The animals with the autografts and 2 of the allografts were untreated, while the animals with the other 2 allografts were treated with azathioprine and prednisolone. The grafts were followed with daily sequential, still photographic recording of the spreading of the fluorescence over the graft surfaces after fluorescein sodium had been given intravenously. A motordriven camera, with automatically timed exposures at very short intervals down to 1 sec, was used in combination with a fast recycling flash unit. The behaviour of the circulation seemed to be the same in auto- and in allografts during the first 8 days of healing. One day before showing signs of rejection at oridinary inspection, the two untreated allografts, on photographic analysis of the spreading of fluorescence, showed minute areas of non-fluorescence in an early stage of the procedure. The same type of local impairment of the circulation, manifested by slower uptake of dye in limited areas, was noted in the treated allografts, appearing 2-3 days before severe signs of rejection were noted at orinary inspection. These areas, however, gradually filled with dye during the further course of the test, to such a degree that the irregularities in the spreading of the fluorescence might easily have been overlooked in an oridinary fluorescein test. The disturbance of the circulation gradually increased during the course of the rejection and, at the stage when changes were observed at ordinary inspection, the circulation was severely impaired, as indicated by a complete non-appearance of fluorescence in certain areas, or in the whole graft. It is suggested that the modification of the classic fluorescein test described here, in detecting the early circulatory irregularities, indicates more precisely the start of the rejection process than do the criteria of allograft rejection used so far, and that it is, therefore, a sensitive means of evaluating the effects of various immunosuppressive procedures.

Animals↗
PubMed1986

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.

Adult↗
PubMed1984

Comparison between fluorescein angiography and fluorescein microscopy.

Fluorescein angiography (FA), which implies that the tissue is reproduced in natural size, and fluorescence microscopy was compared in anesthetized normovolemic and hypovolemic rats in order to define the microscopic background to the fluorescence pattern obtained by FA. It was found that the fluorescence detected by FA by the use of sodium fluorescein as an indicator substance was emitted from the interstitial and intracellular compartments, including the intranuclear. Microscopy showed that sodium fluorescein was located extravascularly in the small intestine within 4 seconds after an intravenous injection. There was a 4 second interval between corresponding microscopic and macroscopic fluorescence pattern.

Angiography↗