Simultaneous quantification of selected glycosaminoglycans by butanolysis-based derivatization and LC-SRM/MS analysis for assessing glycocalyx disruption in vitro and in vivo.

Glycosaminoglycans (GAGs) constitute the main building blocks of the endothelial glycocalyx (GLX), and disruption of GLX initiates and promotes endothelial dysfunction. Here, we aimed to develop a novel, specific and accurate LC-SRM/MS-based method for glycosaminoglycans (GAGs) profiling. The method involved butanolysis derivatization to facilitate GAG-specific disaccharide generation and its subsequent retention in LC–reversed-phase mode followed by mass spectrometric detection performed in positive ion-selected reaction monitoring (SRM) mode. GAG contents were measured in media of endothelial cells (EA.hy926) subjected to various GAG-degrading enzymes, as well as in murine plasma and urine in apolipoprotein E/low‐density lipoprotein receptor‐deficient (ApoE/LDLR −/−) mice and age-matched wild-type C57BL/6 mice. Alternatively, GLX disruption was verified by atomic force microscopy (AFM)-based analysis of GLX thickness. The proposed assay to quantify GAG-specific disaccharides presented high sensitivity for each of the analytes (LLOQ: 0.05–0.1 μg/mL) as well as accuracy and precision (86.8–114.9% and 2.0–14.3%, respectively). In medium of EA.hy926 cells subjected to GAG-degrading enzymes various GAG-specific disaccharides indicating the degradation of keratan sulphate (KS), heparan sulphate (HS), chondroitin sulphate (CHS) or hyaluronan (HA) were detected as predicted based on the characteristics of individual enzyme activity. In turn, AFM-based assessment of GLX thickness was reduced to a similar extent by all single enzyme treatments, whereas the most prominent reduction of GLX thickness was detected following the enzyme mixture. Plasma measurements of GAGs revealed age- and hypercholesterolemia-dependent decrease in GAGs concentration. In summary, a novel LC-SRM/MS-based method for GAG profiling was proposed that may inform on GLX status in cell culture for both in vitro and in vivo conditions. [Display omitted] • A novel LC-SRM/MS method for quantifying GAG-specific disaccharides was proposed. • The method was successfully applied to assess GAG release in in vitro and in vivo models. • The GAG release pattern in endothelial cells in vitro subjected to various enzymes disrupting GLX was characterized. • Evident changes in GAG pattern in plasma related to age- and hypercholesterolemia-dependent endothelial dysfunction were observed. [ABSTRACT FROM AUTHOR]