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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