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-1β) 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-1β. Corrected brain concentrations (Cbr), 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 Cbr 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.

Comparative assessment of in vitro BBB tight junction integrity following exposure to cigarette smoke and e-cigarette vapor: a quantitative evaluation of the protective effects of metformin using small-molecular-weight paracellular markers

“…Sample preparation steps and LC–MS/MS-based detection of stable isotopes-labeled sucrose and mannitol were performed based on the previously validated method [ 49 ]. Briefly, collected samples were diluted in LC–MS/MS grade water in the standard curve range (2–1000 ng/mL).…”

“…For this purpose, sucrose [ 13 C 12 ] and mannitol [ 13 C 6 ] were selected to carry on the permeability studies. We utilized a robust LC–MS/MS method that allows for the simultaneous detection of these tracers with high accuracy and reproducibility [ 49 ].…”

A Quasi-Physiological Microfluidic Blood-Brain Barrier Model for Brain Permeability Studies

“…For this purpose, 100 µg/mL of [ 13 C 6 ] mannitol and [ 13 C 12 ] sucrose (Omicron Biochemicals, South Bend, IN, USA) was added simultaneously to the vascular (lumen) channel. Samples were collected over specific time intervals from the apical channel (ablumen) and measured using an established UPLC-MS/MS method [23]. Briefly, the collected samples were diluted in LC-MS/MS-grade water in the standard curve range (2-1000 ng/mL).…”

“…As such, it is essential to use small-molecular-weight markers to validate the barrier function in these advanced BBB models. We have previously demonstrated the use of an LCMS method using sucrose (353 Da) and mannitol (187 Da) as superior alternatives for BBB permeability studies [23,24]. They possess high metabolic stability, do not interact with proteins, and are uncharged, enabling precise paracellular BBB permeability measurement [23][24][25][26][27].…”

Understanding the brain uptake and permeability of small molecules through the BBB: A technical overview

“…This technique has been further updated recently to allow simultaneous measurements of brain permeability to sucrose as well as mannitol. 115 The importance of BBB permeability measurements in different neurological disorders Over the years numerous compounds and macromolecules have been utilized as markers for elucidating BBB integrity. BBB dysfunction is an important feature of the pathological cascades associated with different neurological disorders such as Alzheimer's disease, 116,117 stroke, 118,119 traumatic brain injury, 120,121 multiple sclerosis, 122,123 and even while anesthetized with volatile anesthetics.…”