LC-MS/MS-based in vitro and in vivo investigation of blood-brain barrier integrity by simultaneous quantitation of mannitol and sucrose.

ABSTRACT:

Background

Understanding the pathophysiology of the blood brain-barrier (BBB) plays a critical role in diagnosis and treatment of disease conditions. Applying a sensitive and specific LC-MS/MS technique for the measurement of BBB integrity with high precision, we have recently introduced non-radioactive [¹³C₁₂]sucrose as a superior marker substance. Comparison of permeability markers with different molecular weight, but otherwise similar physicochemical properties, can provide insights into the uptake mechanism at the BBB. Mannitol is a small hydrophilic, uncharged molecule that is half the size of sucrose. Previously only radioactive [³H]mannitol or [¹⁴C]mannitol has been used to measure BBB integrity.

Methods

We developed a UPLC-MS/MS method for simultaneous analysis of stable isotope-labeled sucrose and mannitol. The in vivo BBB permeability of [¹³C₆]mannitol and [¹³C₁₂]sucrose was measured in mice, using [¹³C₆]sucrose as a vascular marker to correct for brain intravascular content. Moreover, a Transwell model with induced pluripotent stem cell-derived brain endothelial cells was used to measure the permeability coefficient of sucrose and mannitol in vitro both under control and compromised (in the presence of IL-1?) conditions.

Results

We found low permeability values for both mannitol and sucrose in vitro (permeability coefficients of 4.99?±?0.152?×?10^-7 and 3.12?±?0.176?×?10^-7 cm/s, respectively) and in vivo (PS products of 0.267?±?0.021 and 0.126?±?0.025 µl g^-1 min^-1, respectively). Further, the in vitro permeability of both markers substantially increased in the presence of IL-1?. Corrected brain concentrations (C_br), obtained by washout vs. vascular marker correction, were not significantly different for either mannitol (0.071?±?0.007 and 0.065?±?0.009 percent injected dose per g) or sucrose (0.035?±?0.003 and 0.037?±?0.005 percent injected dose per g). These data also indicate that C_br and PS product values of mannitol were about twice the corresponding values of sucrose.

Conclusions

We established a highly sensitive, specific and reproducible approach to simultaneously measure the BBB permeability of two classical low molecular weight, hydrophilic markers in a stable isotope labeled format. This method is now available as a tool to quantify BBB permeability in vitro and in vivo in different disease models, as well as for monitoring treatment outcomes.

SUBMITTER: Noorani B 

PROVIDER: S-EPMC7556948 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature