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 [ 13 C 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 [ 3 H]mannitol or [ 14 C]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 [ 13 C 6 ]mannitol and [ 13 C 12 ]sucrose was measured in mice, using [ 13 C 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-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 (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.

中文翻译：

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通过同时定量甘露醇和蔗糖进行基于 LC-MS/MS 的体外和体内血脑屏障完整性研究

背景 了解血脑屏障 (BBB) 的病理生理学在疾病的诊断和治疗中起着至关重要的作用。应用灵敏且特异的 LC-MS/MS 技术以高精度测量 BBB 完整性，我们最近推出了非放射性 [ 13 C 12 ] 蔗糖作为一种优良的标记物质。比较具有不同分子量但物理化学性质相似的渗透性标记物，可以深入了解 BBB 的吸收机制。甘露醇是一种小的亲水性、不带电荷的分子，大小只有蔗糖的一半。以前只有放射性 [ 3 H] 甘露醇或 [ 14 C] 甘露醇已被用于测量 BBB 的完整性。方法 我们开发了一种 UPLC-MS/MS 方法，用于同时分析稳定同位素标记的蔗糖和甘露醇。在小鼠中测量[ 13 C 6 ]甘露醇和[ 13 C 12 ]蔗糖的体内血脑屏障通透性，使用[ 13 C 6 ]蔗糖作为血管标记物来校正脑血管内容物。此外，具有诱导多能干细胞衍生的脑内皮细胞的 Transwell 模型用于测量体外蔗糖和甘露醇在受控和受损（存在 IL-1β 的情况下）条件下的渗透系数。结果我们发现甘露醇和蔗糖在体外（渗透系数分别为 4.99 ± 0.152 × 10 -7 和 3.12 ± 0.176 × 10 -7 cm/s）和体内（PS 产品为 0.267 ± 0.021 和 0.126 ± 0.025 µl g -1 min -1 ）。此外，两种标志物的体外渗透性在 IL-1β 存在下显着增加。校正的大脑浓度（C br ），对于甘露醇（0.071 ± 0.007 和 0.065 ± 0.009% 注射剂量每克）或蔗糖（0.035 ± 0.003 和 0.037 ± 0.005% 注射剂量每克），通过冲洗与血管标记校正获得的无显着差异。这些数据还表明甘露醇的 C br 和 PS 产品值大约是蔗糖相应值的两倍。结论 我们建立了一种高度灵敏、特异性和可重复的方法，以稳定同位素标记形式同时测量两种经典低分子量、亲水性标记物的 BBB 渗透性。该方法现在可用作量化不同疾病模型中体外和体内 BBB 通透性的工具，以及用于监测治疗结果。007 和 0.065 ± 0.009% 注射剂量/g）或蔗糖（0.035 ± 0.003 和 0.037 ± 0.005% 注射剂量/g）。这些数据还表明甘露醇的 C br 和 PS 产品值大约是蔗糖相应值的两倍。结论 我们建立了一种高度灵敏、特异且可重复的方法，以稳定同位素标记形式同时测量两种经典低分子量亲水性标记物的 BBB 渗透性。该方法现在可用作量化不同疾病模型中体外和体内 BBB 通透性的工具，以及用于监测治疗结果。007 和 0.065 ± 0.009% 注射剂量/g）或蔗糖（0.035 ± 0.003 和 0.037 ± 0.005% 注射剂量/g）。这些数据还表明甘露醇的 C br 和 PS 产品值大约是蔗糖相应值的两倍。结论 我们建立了一种高度灵敏、特异且可重复的方法，以稳定同位素标记形式同时测量两种经典低分子量亲水性标记物的 BBB 渗透性。该方法现在可用作量化不同疾病模型中体外和体内 BBB 通透性的工具，以及用于监测治疗结果。这些数据还表明甘露醇的 C br 和 PS 产品值大约是蔗糖相应值的两倍。结论 我们建立了一种高度灵敏、特异且可重复的方法，以稳定同位素标记形式同时测量两种经典低分子量亲水性标记物的 BBB 渗透性。该方法现在可用作量化不同疾病模型中体外和体内 BBB 通透性的工具，以及用于监测治疗结果。这些数据还表明甘露醇的 C br 和 PS 产品值大约是蔗糖相应值的两倍。结论 我们建立了一种高度灵敏、特异且可重复的方法，以稳定同位素标记形式同时测量两种经典低分子量亲水性标记物的 BBB 渗透性。该方法现在可用作量化不同疾病模型中体外和体内 BBB 通透性的工具，以及用于监测治疗结果。