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 [C-13(12)]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 [H-3]mannitol or [C-14]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 [C-13(6)]mannitol and [C-13(12)]sucrose was measured in mice, using [C-13(6)]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 beta) conditions. Results We found low permeability values for both mannitol and sucrose in vitro (permeability coefficients of 4.99 +/- 0.152 x 10(-7)and 3.12 +/- 0.176 x 10(-7)cm/s, respectively) and in vivo (PS products of 0.267 +/- 0.021 and 0.126 +/- 0.025 mu l g(-1)min(-1), respectively). Further, the in vitro permeability of both markers substantially increased in the presence of IL-1 beta. 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.