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Table 2 Summary of different markers that can be used for BBB permeability studies

From: A blood–brain barrier overview on structure, function, impairment, and biomarkers of integrity

Marker Size (Da) Binding Advantage Disadvantage
Protein Tissue
Radiolabeled-mannitol 182 No No No interaction with proteins
Metabolically stable
Uncharged
No interaction with BBB transporters
Suitable for small molecules permeability prediction
Contains lipophilic impurities
Requires a radioactive license
High cost
It cannot be visualized
Biotin ethylenediamine 286 No No It can be measured quantitatively with HPLC
Visual qualification is feasible
It has a low binding to plasma proteins
Radiolabeled-sucrose 342 No No No interaction with proteins
Metabolically stable
Uncharged
No interactions with BBB transporters
Suitable for small molecules permeability prediction
Contains lipophilic impurities
Over-time degradation
Requires a radioactive license
High cost
It cannot be visualized
13C12 sucrose 354 No No Non-radioactive
A sensitive and specific method of detection
No interaction with BBB transporters
Metabolically stable
No interaction with protein
Suitable for small molecules permeability prediction
Requires LC–MS/MS device for detection
High cost
It cannot be visualized
Sodium fluorescein 376 Weak NR Easy detection method
Freely diffusible
Detectable in very low concentrations
Inexpensive
Non-radioactive
Nontoxic
It can be visually assessed
Suitable for small molecules permeability prediction
Interaction with BBB transporters
Weekly binds to plasma proteins
Evans blue 961 Yes Yes Visual investigation and qualification are feasible using different microscope techniques
Allow assessment of vascular permeability to macromolecules due to binding to its extensive binding to albumin
Quantification is unreliable
It strongly binds to serum albumin in vivo and in vitro and, thereby, becomes a high molecular weight protein tracer (69 kDa)
Potential in vivo toxicity
Not suitable for small molecules permeability prediction
Trypan blue 961 Yes Yes Visual evaluation is feasible Quantification is unreliable
Binds to plasma proteins
Not suitable for small molecules permeability prediction
Radio-inulin 7000 No No Quantification is feasible
No protein binding
Contains lipophilic impurities
Requires a radioactive license
High cost
It cannot be visualized
Horseradish peroxidase 44,000 NR NR Can be easily visualized under light and electron microscopy The possibility of diffusion artifacts resulted in a distribution of the reaction product that may not reflect actual protein
Found to be toxic in large doses
Not suitable for small molecules permeability prediction
Quantification is unreliable
Species-specific degranulation of mast cells and histamine release
Albumin 69,000 No No It is widely used in the radiolabeled or fluorescently labeled form
The fluorescent-labeled version could be used for morphological studies
The radiolabeled version allows for accurate quantification
Requires radioactive license
Not suitable for small molecules permeability prediction
Dextrans 1500 to 70,000 No No It can be used for a broad range of molecular weights
Can be visualized with both light microscopic and electron microscopic level due to conjugation with biotin of FITC
Not suitable for small molecules permeability prediction
Stability issue
May be toxic at high concentrations
  1. NR not reported
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