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PubMed1991

Carpopedal spasm: an unusual reaction to fluorescein?

Fluorescein angiography is a routine procedure in ophthalmology. There can, however, be some harmful side-effects from fluorescein injections. It is important that all personnel involved in using this investigative procedure should be aware at all times of the possible side-effects and be ready to detect and control any adverse reaction. A case presentation is given of a 40-year-old man who had an unusual reaction to fluorescein resulting in carpopedal spasm.

Adult↗
PubMed1975

Excitation and emission spectra of fluorescein dye in the human ocular fundus.

The excitation and emission spectra of fluorescein dye were determined during angiography from different sites in the human ocular fundus. All spectra were markedly shifted toward longer wavelengths relative to the spectra of fluorescein in aqueous solution. This effect is most pronounced for the macular area; however, it decreases for the choroidal background and even more for the retinal vessels. The results are relevant to the selection of optical filter combinations for fluorescein angiography.

Adolescent↗
PubMed1992

Activation and inhibition of ATP-sensitive K+ channels by fluorescein derivatives.

Fluorescein derivatives are known to bind to nucleotide-binding sites on transport ATPases. In this study, they have been used as ligands to nucleotide-binding sites on ATP-sensitive K+ channels in insulinoma cells. Their effect on channel activity has been studied using 86Rb+ efflux and patch-clamp techniques. Fluorescein derivatives have two opposite effects. First, like ATP, they can inhibit active ATP-sensitive K+ channels. Second, they are able to reactivate ATP-sensitive K+ channels subjected to inactivation or "run-down" in the absence of cytoplasmic ATP. Therefore reactivation of the inactivated ATP-sensitive K+ channel clearly does not require channel phosphorylation as is commonly believed. The results indicate the existence of two binding sites for nucleotides, one activator site and one inhibitor site. Irreversible binding at either the inhibitor or the activator site on the channel was obtained with eosin-5-maleimide, resulting in irreversible inhibition or activation of the ATP-sensitive K+ channel respectively. The irreversibly activated channel could still be inhibited by 2 mM ATP. After activation by fluorescein derivatives, ATP-sensitive K+ channels become resistant to the classical blocker of this channel, the sulfonylurea glibenclamide. Negative allosteric interactions between fluorescein/nucleotide receptors and sulfonylurea-binding sites were suggested by results obtained in [3H]glibenclamide-binding experiments.

Adenosine Triphosphate↗
PubMed2006

[Fluorescein angiography of retinal neovascularization model in mice].

OBJECTIVE: To investigate a high molecular weight fluorescein angiography in the application of retinal neovascularization model in mouse. METHODS: retinal neovascularization model was induced by exposure mouse to an environment containing high concentration of oxygen. High molecular weight fluorescein isothiocyanate dextran were perfused through the left ventricle directly, then the mouse eyes were enucleated and fixed with 4% paraformaldehyde. The retina was separated from the eyecup and flat mounting was performed on a gelatin coated slide. The vasculature was examined under fluorescent microscope. RESULT: The whole retinal vasculature was clearly visualized under fluorescent microscope. By focusing on different layer of the tissue, superficial, deep vascular plexus and connecting vessels also could be distinguished. The neovascular response occurred at the junction between the vascular and the avascular retina. CONCLUSION: High molecular weight fluorescein angiography can be applied for retinal neovascularization evaluation.

Animals↗
PubMed1991

Single-chain site-specific mutations of fluorescein-amino acid contact residues in high affinity monoclonal antibody 4-4-20.

Previous crystallographic studies of high affinity anti-fluorescein monoclonal antibody 4-4-20 (Ka = 1.7 x 10(10) M-1) complexed with fluorescyl ligand resolved active site contact residues involved in binding. For better definition of the relative roles of three light chain antigen contact residues (L27dhis, L32tyr and L34arg), four site-specific mutations (L27dhis to L27lys, L32tyr to L32phe, and L34arg to L34lys and L34his) were generated and expressed in single-chain antigen binding derivatives of monoclonal antibody 4-4-20 containing two different polypeptide linkers (SCA 4-4-20/205c, 25 amino acids and SCA 4-4-20/212, 14 amino acids). Results showed that L27dhis and L32tyr were necessary for wild type binding affinities, however, were not required for near-wild type Qmax values (where Qmax is the maximum fluoroscein fluorescence quenching expressed as percent). Tyrosine L32 which hydrogen bonds with ligand was also characterized at the haptenic level through the use of 9-hydroxyphenylfluoron which lacks the carboxyl group to which L32 tyrosine forms a hydrogen bond. Results demonstrated that wild type SCA and mutant L32phe possessed similar HPF binding characteristics. Active site contact residue L34arg was important for fluorescein quenching maxima and binding affinity (L34his mutant), however, substitution of lysine for arginine at L34 did not have a significant effect on observed Qmax value. In addition, substitutions had no effect on structural and topological characteristics, since all mutants retained similar idiotypic and metatypic properties. Finally, two linkers were comparatively examined to determine relative contributions to mutant binding properties and stability. No linker effects were observed. Collectively, these results verified the importance of these light chain fluorescein contact residues in the binding pocket of monoclonal antibody 4-4-20.

Amino Acid Sequence↗
PubMed1990

Active site structure and antigen binding properties of idiotypically cross-reactive anti-fluorescein monoclonal antibodies.

This report includes complete VH and V kappa nucleotide and deduced amino acid sequences of idiotypically cross-reactive monoclonal anti-fluorescein antibodies that differed greater than 10(5)-fold in affinity. High affinity monoclonal antibody 4-4-20 and intermediate affinity antibodies 10-25, 5-14, 9-40, 12-40, and 3-24 utilized greater than or equal to 90% homologous VHIIIC germ-line genes. Extensive D segment length and sequence variability were observed; however, compensatory germ-line JH4 (4-4-20 and 3-24) or JH3 (10-25, 5-14, 9-40, and 12-40) sequence lengths resulted in H chain CDR3 + FR4 to be a constant 18 amino acids. In addition, each antibody and low affinity 3-13 rearranged greater than or equal to 96% homologous V kappa II genes to J kappa 1, except for 10-25 (J kappa 5) and 3-13 (J kappa 4). Resolved crystal structure of complexed fluorescein and 4-4-20 Fab fragments revealed residues HisL27d, TyrL32, ArgL34, SerL91, TrpL96, and TrpH33 acted as hapten contact residues. Antibodies 5-14, 9-40, 12-40, and 3-24 primary structures possessed identical contact residues as 4-4-20 except for the substitution of HisL34 for ArgL34. Thus, ArgL34 was implicated in the increased affinity of monoclonal antibody 4-4-20. Finally, it was difficult to correlate extensive H chain CDR3 residue heterogeneity directly with fluorescein binding and idiotypy.

Amino Acid Sequence↗
PubMed1989

Flow cytometric analysis of glucocorticoid receptor using monoclonal antibody and fluoresceinated ligand probes.

Conditions were established for single cell analysis of glucocorticoid receptor (GR) content by flow cytometry using several clones of a human leukemic cell line (CCRF-CEM). These included CEM-7A, 7R, C1, and ICR 27 Tk.3 cells which were examined both by standard [3H]dexamethasone radiometric binding and by two independent flow cytometry assays. The latter involved either mouse monoclonal antibody against GR (GR-MoAb) or fluoresceinated cortisol ligand probes. For CEM-7A, 7R, and C1 cells, there was a correlation between GR-MoAb and radiometrically defined GR values. However, clone ICR-27 Tk.3 with low [3H]dexamethasone binding exhibited the highest GR-MoAb fluorescence. The fluoresceinated cortisol assay correlated with dexamethasone binding values in all four clones. Thus, GR-MoAb identifies the total immunologically reactive GR present, while the fluoresceinated cortisol assay quantifies only the functionally intact GR in terms of its initial binding. Their combined use may reveal the cellular heterogeneity of GR expression and function also in human tumor samples, to which they have been successfully applied. When coupled with DNA counterstaining, GR expression can be related directly to frequently DNA-aneuploid tumor cells and cell cycle distribution.

Antibodies, Monoclonal↗
PubMed1988

Detection thresholds of nonocclusive intestinal hypoperfusion by Doppler ultrasound, photoplethysmography, and fluorescein.

Because clinical assessment of bowel viability is unreliable, other methods of determining intestinal perfusion have been recommended. Since none of these quantifies intestinal blood flow, we measured flow at the detection thresholds of Doppler ultrasound, photoplethysmography, and intravenously administered fluorescein, perfused the intestines at these threshold levels, and assessed histologic evidence of ischemic damage. The intestines of five anesthetized dogs were perfused for 4 hours via an in-line pulsatile extra-corporeal circuit assembled between the iliac and superior mesenteric arteries at either relatively physiologic (approximately 20 ml/min/kg body weight) levels or reduced levels representing the flow detection thresholds of Doppler ultrasound or photoplethysmographic probes (approximately 4 ml/min/kg). Intravenously administered fluorescein was detected at even lower perfusion levels (approximately 2.1 ml/min/kg). Clear-cut ischemic changes were documented histologically in all subjects perfused at Doppler/PPG flow detection thresholds but in none of those perfused at normal levels. We conclude that threshold blood flow detection by any one of these methods, especially fluorescein, occurs at levels inadequate to guarantee tissue viability.

Animals↗
PubMed1986

Conformational transitions in fluorescein-labeled (Na,K)ATPase reconstituted into phospholipid vesicles.

Fluorescein-labeled (Na,K)ATPase reconstituted into phospholipid vesicles has been used to study conformational transitions. Addition of K+ or Na+ to the vesicle medium induces fluorescence changes characteristic of the E2(K) or E1Na states of fluorescein-labeled (Na,K)ATPase (Karlish, S.J.D. (1980) J. Bioenerg. Biomembr. 12, 111-136). The cation effects are exerted from the cytoplasmic surface of inside-out-oriented pumps. Equilibrium cation titrations and measurements of rates of conformational transitions have led to the following observations. 1) The rate of E2(K)----E1Na or E2(T1)----E1Na is 4-6-fold faster and E1K----E2(K) is about 2-fold slower in vesicles compared to enzyme. In equilibrium titrations the K0.5 for K+ is higher and that for Na+ is lower for vesicles compared to enzyme. The conformational equilibrium E(1)2K----E2(2K) is apparently shifted toward E(1)2K in vesicles compared to enzyme. 2) Diffusion potentials, positive-outside, induced with valinomycin or Li+ ionophore AS701, do not affect the rates of E2(T1)----E1Na or E1K----E2(K), or equilibrium cation titrations. This demonstrates that the conformational transitions E(1)2K----E2(2K) are voltage-insensitive steps, confirming a prediction based on transport experiments. 3) In vesicles containing choline, K+, Na+, or Li+, the rate of E2(T1)----E1Na increases in the order given. Vesicles with reconstituted fluorescein-labeled (Na,K)ATPase provide a convenient system for correlating directly properties of conformational transitions with cation transport.

Animals↗
PubMed1987

Skin necrosis following fluorescein extravasation. A survey of the Macula Society.

Three patients developed cellulitis and skin necrosis following fluorescein dye extravasation. This experience prompted a survey of the Macula Society membership to determine whether this complication of fluorescein angiography is as rare as the paucity of cases in the literature suggests. In addition, the manufacturer was asked to reexamine the dye lot to determine whether an impurity had been introduced unintentionally during the manufacturing process. No impurity was found in the dye lots tested. The survey disclosed only nine additional cases of skin necrosis, leading to the conclusion that skin necrosis probably represents a rare, idiosyncratic reaction. Although fluorescein angiography remains a safe procedure, efforts should be directed toward prevention of dye extravasation. When extravasation does occur, prompt and proper medical attention with close follow-up study may minimize the likelihood of skin necrosis.

Aged↗
PubMed1985

The analysis of an anti-fluorescein response at limit dilution: the effect of lymphokine preparations on the apparent specificity.

The specificity of cytotoxic T cells in an anti-fluorescein response has been analysed at limit dilution. According to the ability to discriminate between fluoresceinated and non-fluoresceinated targets, the apparent specificity of CTL depends on the quality and quantity of the lymphokine preparations used to support the response. An acid-labile component of the factor preparations contributes to the generation of non-specific CTL. No cytotoxicity against NK-sensitive target cells was detected in the 'non-specific' component of the responses.

Acids↗
PubMed1985

The effect of pH on the transfer of fluorescein across the blood-retinal barrier.

The blood-retinal barrier (BRB) might be governed by the same permeability principles as the blood-brain barrier (BBB). For a weak acid like fluorescein, BRB permeability would be proportional to its pH-dependent lipid solubility, according to the pH partition hypothesis. A range of metabolic acidosis was produced in 20 rats by the oral administration of NH4Cl; six additional rats received normal saline. Four hours later, vitreous fluorophotometry, venous fluorescein values, and arterial pH were measured. Significant linear relationships were found between vitreous fluorophotometry readings and blood hydrogen ion concentrations (p less than 0.025) and plasma fluorescein concentrations (p less than 0.05). According to the linear relationship, changing the pH from 7.4 to 7.3 or 6.9 would result in an increase in vitreous fluorophotometry reading of 8.5 or 72%, respectively. Since the pH partition hypothesis predicts values of 52 or 640% our results suggest that the BRB conforms less to the hypothesis than does the BBB. Furthermore, although pH changes of a magnitude able to influence vitreous fluorophotometry readings substantially may occur under experimental conditions in animals, they are unlikely to occur in ambulatory human patients.

Animals↗
PubMedDate not supplied

[Measurements of the potential difference during adsorption of phloretin and fluorescein on the surface of lipid membranes using the method of inner field compensation].

The inner field compensation method was used for the measurement of the boundary dipole potentials as a function of phloretin and fluorescein concentration, ionic strength and pH. These potentials were compared with calculated from the conductance change in the presence of nonactin. Both methods gave similar results for fluorescein, but the steady-state potentials in the case of phloretin, obtained by the first method were smaller. These results imply that fluorescein, being introduced on one side of the BLM, remained on that side contrary to phloretin which penetrated through the membrane and partitioned between its both boundaries.

Adsorption↗
PubMed1984

[Results of fluorescein nose endoscopy in the diagnosis of cerebrospinal rhinorrhea].

To establish the diagnosis of cerebrospinal rhinorrhoea 45 patients were examined via intrathecal fluorescein-dye studies. Rhinorrhoea was suspected after head injuries and surgery of the base of the skull. The diagnostic procedure was in accordance with the technique of Messerklinger. In 8 patients a fluorescent secretion was detected and a CSF-leakage operatively proven. The result of the fluorescein tests, the x-ray tomographies and the RIHSA-studies are compared. The fluorescein technique is shown to be a useful diagnostic tool in the diagnosis of cerebrospinal leaks.

Brain Injuries↗
PubMed1983

Adverse reactions in intravenous fluorescein angiography.

Adverse reactions were noted in 241 (4.82%) of 5,000 consecutive intravenous fluorescein angiographies of the retina. The most frequent adverse reactions were nausea (2.24%), vomiting (1.78%), and urticaria/pruritus (0.34%). No life-threatening reactions were noted. No significant difference in the rate of adverse reactions was found when angiography using 10% fluorescein was compared with angiography using 25% fluorescein.

Female↗
PubMed1983

Kinetics of intraocular penetration of topical fluorescein: analysis by new method.

The kinetics of the intraocular penetration of topically applied fluorescein was analyzed by a new method. To the time-courses of fluorescein concentration changes in the aqueous, the bi-exponential equation of Jones-Maurice for a two-compartment model was fitted by a method of least squares. The initial amount of the dye in the cornea divided by the anterior chamber volume, Mo/Va, the cornea-aqueous transfer coefficient in reference to the corneal volume, Kc.ca, and the loss rate of the dye from the anterior chamber, ko, were treated as unknown independent parameters, and they were estimated directly from the concentration changes in the anterior chamber. In the instillation experiments, 20 microliter of phosphate buffered 30% fluorescein solution was instilled in both eyes of 11 normal young subjects and the eyes were washed thoroughly after 5 minutes. The following values were obtained: kc.ca: 4.8 +/- 0.5 X 10(-3) min-1 (Mean +/- SD), ko: 0.78 +/- 0.09 X 10(-2) min-1, and Mo/Va: 0.20 +/- 0.25 micrograms ml-1. The right-left ratio averaged 1.01 +/- 0.05 in kc.ca and 1.02 +/- 0.05 in ko. A similar analysis was carried out in 12 normal young subjects, where the dye was administered to the cornea by ionotophoresis and the results were as follows: kc.ca: 4.7 +/- 0.6 X 10(-3) min-1, ko: 0.83 +/- 0.19 X 10(-2) min-1, and Mo/Va: 0.15 +/- 0.16 micrograms ml-1. The right-left ratio averaged 0.93 +/- 0.12 in kc.ca and 1.02 +/- 0.18 in ko. The anterior chamber volume, Va, was determined by the method of Johnson et al. and it averaged 202 microliter. The aqueous flow rate, i.e., ko.Va, was 1.61 microliter min-1.

Administration, Topical↗
PubMed1982

Site-specific fluorescein-labeled cobra alpha-toxin. Biochemical and spectroscopic characterization.

Cobra alpha-toxin purified from Naja naja siamensis venom was labeled with near stoichiometric quantities of fluorescein isothiocyanate. A monofluorescein alpha-toxin was separated in 50-60% yield from unconjugated alpha-toxin and other reaction products by ion exchange chromatography. The isolated mono-conjugated alpha-toxin electrofocuses largely as a single entity with 92% appearing with a pI of 9.6. The unmodified toxin has a pI of 10.7. Thermolysin digestion and subsequent high pressure liquid chromatography of the peptides yield two dominant fluorescent peaks, both of which can be traced to the labeling of lysine 23. The NE-fluorescein isothiocyanate (FITC)-Lys-23 alpha-toxin shows an apparent reduction in quantum yield when compared with either free FITC or the denatured and reduced NE-FITC-Lys-23 alpha-toxin. The reduction of fluorescence is likely to be due to static quenching of the fluorescein by the tryptophanyl and tyrosyl residues (25 and 21, respectively) in the "central loop" region. Binding of the NE-FITC-Lys-23 alpha-toxin to the membrane-associated acetylcholine receptor is accompanied by a 95 +/- 22% increase in fluorescence which probably reflects perturbation of the beta-pleated sheet character of the region containing residues 20-25 of the alpha-toxin. Steady state fluorescence polarization measurements of NE-FITC-Lys-23 alpha-toxin yield a rotational correlation time of 3.7 ns, suggesting FITC is largely immobilized on the alpha-toxin. The NE-FITC-Lys-23 alpha-toxin binds with a dissociation constant of 4 nM determined by fluorescence polarization.

Amino Acids↗
PubMed1978

Anterior segment permeability to fluorescein-labeled dextrans in the rat.

Permeability of the anterior segment of adult Wistar-Furth rats to fluorescein-labeled dextrans (FITC-dextrans) was evaluated by fluorescein angiography following intravenous injection of 0.2 ml of a 33% FITC-dextran solution. Permeation of each fluorescent compound into the anterior chamber was evaluated by (1) initial appearance of fluorescence in iris vessels, (2) the appearance of fluorescence at the pupil, (3) contrast between the iris vessels and their surroundings, and (4) disappearance of FITC-dextrans from the iris blood column. Permeability of FITC-dex 3 (molecular weight, 3000; effective diffusion radius, 12 A) was similar to that of sodium fluorescein in that both entered the aqueous rapidly and showed diffuse aqueous fluorescence within 3 min. As the molecular weight of the FITC-dextrans increased (m.w. of 20,000, 40,000, 70,000, and 150,000 were used), the FITC-dextrans exhibited decreased aqueous permeation, and iris vessel contrast persisted. For FITC-dex 150 (m.w. 150,000; 85 A) no fluorescence was visible in the pupil at times as long as 1 hr. Since these fluorescent compounds exhibit aqueous permeability properties based upon molecular size and thus are similar to blood proteins, they should be useful for in vivo and in vitro quantitative study of the blood-aqueous barrier in animals.

Animals↗