Fluorescein is a synthetic organic photoactive dye molecule that dissolves in water, alcohol, and solvents [1].

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- Peroxidase-mimetic activity: Fluorescein exhibited peroxidase-like catalytic activity, capable of catalyzing the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H₂O₂) to form a blue-colored oxidized product. The catalytic reaction followed Michaelis-Menten kinetics with a Km value of 0.21 mM for TMB and 1.85 mM for H₂O₂, and a Vmax of 5.76 × 10⁻⁸ M·s⁻¹. The optimal pH for the catalytic activity was 4.0, and the activity was maintained at 80% of the maximum when incubated at 40°C for 1 hour [3]
- Antimicrobial activity: Fluorescein alone showed weak antimicrobial activity against Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) with inhibition zone diameters ranging from 6.2 to 7.5 mm at a concentration of 100 μg/mL. In contrast, its nanohybrids with metal oxides exhibited enhanced antimicrobial efficacy [1]

- Peroxidase-mimetic activity assay: A reaction mixture was prepared by mixing Fluorescein (final concentration 0.1 mg/mL), TMB (final concentration 0.5 mM), and H₂O₂ (final concentration 1 mM) in acetate buffer (pH 4.0). The mixture was incubated at 37°C for 30 minutes, and the formation of oxidized TMB was monitored by measuring the absorbance at 652 nm. Kinetic parameters (Km, Vmax) were calculated by varying the concentration of TMB (0.1-1.0 mM) or H₂O₂ (0.5-5.0 mM) while keeping the other substrate concentration constant. The thermal stability was evaluated by incubating Fluorescein at different temperatures (20-80°C) for 1 hour before performing the assay [3]

Absorption, Distribution and Excretion
Rapid Distribution
Luciferin and its metabolites are primarily excreted via the kidneys.
0.5 L/kg
Renal clearance = 1.75 mL/min/kg [after intravenous administration]
Hepatic clearance = 1.50 mL/min/kg [after intravenous administration]
Skin fluorescence can persist for several hours, and luciferin can persist in urine for up to 30 hours.
Fluoriferin typically appears in the central ophthalmic artery within 7 to 14 seconds after intravenous injection via the antecubital vein. Following intravenous injection of sodium luciferin, yellow discoloration of the skin occurs within minutes and begins to fade 6 to 12 hours after administration. Various volume of distribution estimates indicate that luciferin distributes well into the interstitial space (0.5 L/kg).
Luciferin and its metabolites are primarily excreted via the kidneys. Following intravenous administration, mild fluorescence persists in urine for 24 to 36 hours. Renal clearance was estimated at 1.75 mL/min/kg, and hepatic clearance (due to binding) at 1.50 mL/min/kg. Systemic clearance of fluorescein was essentially completed within 48 to 72 hours after administration of 500 mg. Fluorescein sodium has been confirmed to be secreted into human milk. The permeability of the blood-retinal barrier and blood-aqueous barrier, as well as aqueous humor flow rate, of fluorescein can be estimated by measuring the concentrations of fluorescein in the vitreous humor, aqueous humor, and plasma after systemic administration. Fluorescein is usually determined by fluorescence methods, but its metabolite, fluorescein glucuronide, is also fluorescent. To evaluate the effect of fluorescein glucuronide on the fluorescence-based quantification of fluorescein, we investigated the pharmacokinetics of fluorescein and its metabolite fluorescein glucuronide in plasma over 38 hours after intravenous administration of 14 mg/kg fluorescein sodium in five healthy subjects. Fluorescein and its metabolite fluorescein glucuronide in plasma and plasma ultrafiltrate were determined using fluorescence methods and high-performance liquid chromatography (HPLC). In our fluorometer, the fluorescence intensity of luciferin glucuronide is 0.124 times that of luciferin. Luciferin glucuronide is rapidly converted to luciferin glucuronide; within 10 minutes, the concentration of free luciferin glucuronide exceeds that of free luciferin. The terminal half-lives of luciferin and luciferin glucuronide in plasma ultrafiltrate are 23.5 minutes and 264 minutes, respectively; therefore, after 4-5 hours, luciferin glucuronide contributes almost all of the plasma fluorescence. Because the binding rate of luciferin glucuronide in plasma is lower than that of luciferin, the fluorescence ratio of plasma ultrafiltrate to plasma increases over time. Shortly after injection, most of the luciferin permeates into the various compartments of the eye. ...

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Metabolism/Metabolites
By measuring the concentration of luciferin in the vitreous humor, aqueous humor, and plasma after systemic administration, the permeability of the blood-retinal barrier and the blood-aqueous humor barrier of luciferin, as well as the flow rate of aqueous humor, can be estimated. Luciferin is typically measured using fluorescence methods, but its metabolite, luciferin glucuronide, also exhibits fluorescence. To evaluate the effect of luciferin glucuronide on the fluorescence-based quantification of luciferin, we investigated the pharmacokinetics of luciferin and its metabolite luciferin glucuronide in plasma over 38 hours following intravenous injection of 14 mg/kg sodium luciferin in five healthy subjects. Luciferin and its metabolite luciferin glucuronide in plasma and plasma ultrafiltrate were determined using fluorescence and high-performance liquid chromatography (HPLC). In our fluorometer, the fluorescence intensity of luciferin glucuronide was 0.124 times that of luciferin. Luciferin glucuronide is rapidly converted to luciferin glucuronide; within 10 minutes, the concentration of free luciferin glucuronide exceeds that of free luciferin. The terminal half-lives of luciferin and luciferin glucuronide in plasma ultrafiltrate are 23.5 minutes and 264 minutes, respectively; therefore, after 4–5 hours, luciferin glucuronide contributes almost all of the plasma fluorescence. Because the binding rate of fluorescein glucuronide in plasma is lower than that of fluorescein itself, the fluorescence ratio of plasma ultrafiltrate to plasma increases over time. Shortly after injection, most of the fluorescein penetrates into the ocular tissues. …
It is rapidly metabolized to fluorescein monoglucuronide. In 7 healthy subjects, after intravenous injection of sodium fluorescein (14 mg/kg), approximately 80% of the fluorescein in plasma was converted to glucuronide conjugates within 1 hour, indicating a relatively rapid binding rate.
Fluorescence is a known human metabolite of zinc.
Elimination pathway: Fluorescein and its metabolites are primarily excreted via the kidneys.
Biological half-life
…The pharmacokinetics of fluorescein and its glucuronide in plasma were studied within 38 hours after intravenous injection of 14 mg/kg sodium fluorescein in 5 healthy subjects. The terminal half-lives of fluorescein and fluorescein glucuronide in plasma ultrafiltrate were 23.5 minutes and 264 minutes, respectively, ...

Toxicity Summary


Sodium fluorescein is a widely used fluorescent compound or fluorophore in ophthalmic diagnosis, with a maximum absorption wavelength of 494 nm and a maximum emission wavelength of 521 nm. The yellow-green fluorescence of this compound can be used to mark and distinguish observed vascular areas from adjacent areas. It can be used topically as eye drops or intravenously for fluorescein angiography. Topical application of fluorescein is an effective tool for diagnosing corneal abrasions, corneal ulcers, herpetic keratitis, and dry eye. Fluorescein angiography is used to diagnose and classify macular degeneration, diabetic retinopathy, inflammatory intraocular diseases, and intraocular tumors.


Use during Pregnancy and Lactation

◉ Overview of Use During Lactation

Due to limited ocular absorption, fluorescein is not expected to have any adverse effects on breastfed infants. After using the eye drops, to significantly reduce the amount of medication entering breast milk, press the tear duct at the corner of the eye for at least 1 minute, then blot away any excess medication with absorbent tissue.
After intravenous administration to a breastfeeding mother, the concentration of the drug in breast milk and the dose ingested by the infant are significantly higher than those for ocular application. These higher levels are unlikely to cause problems for most infants, but the infant should likely avoid exposure to strong light, such as phototherapy, for several days after the mother's administration.
◉ Effects on breastfed infants
No published information found as of the revision date.
◉ Effects on lactation and breast milk
No published information found as of the revision date.

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Protein binding
85%

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Non-human toxicity values
Mouse LD50: Approximately 4738 mg/kg /fluorescein sodium/
Rat LD50: Approximately 6721 mg/kg /fluorescein sodium/

[1]. Fluorescein dye derivatives and their nanohybrids: Synthesis, characterization and antimicrobial activity. J Photochem Photobiol B. 2016 Sep;162:421-433.

[2]. Analysis of chemical equilibrium of silicon-substituted fluorescein and its application to develop a scaffold for red fluorescent probes. Anal Chem. 2015;87(17):9061-9069.

[3]. Fluorescein as an artificial enzyme to mimic peroxidase. Chem Commun (Camb). 2016 Nov24;52(96):13912-13915.

[4]. Absorption and fluorescence properties of fluorescein. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy.

Therapeutic Uses


Contrast Agent

Veterinary Use: Deep corneal ulcers, Descemet's membrane bulging, and iris prolapse are common in dogs, cats, and horses. …Important diagnostic aids include: the Schirmer test to measure tear secretion, and topical application of fluorescein to examine corneal ulcers. …
Fluorescence is currently used to determine circulation time, blood supply adequacy, and tissue viability.
In determining circulation time, …fluorescein is administered via rapid intravenous injection…the appearance of fluorescence in the lips, eyes, or intact skin or wheals (histamine or scratches)…is considered the endpoint.
Measuring circulation time from the arm to the retina is used to diagnose carotid artery occlusion.

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Drug Warnings
Adverse reactions that may occur after topical application to the eyes include irritation and rash. Fluorescein may cause yellowing of the skin or eyes. Urine may be bright yellow. Adverse reactions after intravenous injection include nausea, vomiting, headache, dizziness, syncope, and hypotension.

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- Luciferin exists in pH-dependent equilibrium as lactone, quinone, and zwitterionic forms. Chemical equilibrium: Luciferin exists. The quinone form (responsible for fluorescence) is dominant under neutral to alkaline pH conditions, while the lactone form is dominant under acidic pH conditions [2].
- In aqueous solution (pH 7.0), the maximum absorption wavelength (λmax) is 494 nm and the molar absorptivity (ε) is 76,000 M⁻¹·cm⁻¹. Spectral properties: Luciferin. The maximum fluorescence emission wavelength (λem) is 512 nm and the fluorescence quantum yield (Φ) is 0.92 [4].
- It can be used as a multifunctional scaffold to develop red fluorescent probes by silicon substitution, thereby shifting the absorption and emission wavelengths to a longer range (λmax ~ 530 nm, λem ~ 550 nm) while maintaining a high fluorescence quantum yield. Applications as probe scaffolds: Fluorescence [2] - is a widely used xaton dye, whose derivatives are synthesized by modifying the xaton core or carboxyl group to enhance biological activity or physicochemical properties. Synthetic background: Fluorescence [1] - can function as a small molecule artificial enzyme (nanozyme mimic) due to its ability to mimic the catalytic behavior of natural peroxidases, thanks to its electron-rich aromatic structure, which facilitates... electron transfer between H₂O₂ and substrate. Artificial enzyme background: Fluorescence [3]

C20H12O5

332.3063

332.068

2321-07-5

Fluorescein sodium;518-47-8

16850

Pink to red solid powder

1.6±0.1 g/cm3

620.8±55.0 °C at 760 mmHg

320 °C(lit.)

232.6±25.0 °C

0.0±1.9 mmHg at 25°C

1.792

2.98

2

5

0

25

522

0

GNBHRKFJIUUOQI-UHFFFAOYSA-N

InChI=1S/C20H12O5/c21-11-5-7-15-17(9-11)24-18-10-12(22)6-8-16(18)20(15)14-4-2-1-3-13(14)19(23)25-20/h1-10,21-22H

3',6'-dihydroxyspiro[2-benzofuran-3,9'-xanthene]-1-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~250 mg/mL (~752.31 mM)
H2O : ~1 mg/mL (~3.01 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.26 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.26 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)