Analysis of procainamide-derivatised heparan sulphate disaccharides in biological samples using hydrophilic interaction liquid chromatography mass spectrometry

Glycosaminoglycans (GAGs) are a family of linear heteropolysaccharides made up of repeating disaccharide units that are found on the surface and extracellular matrix of animal cells. They are known to play a critical role in a wide range of cellular processes including proliferation, differentiation and invasion. To elucidate the mechanism of action of these molecules, it is essential to quantify their disaccharide composition. Analytical methods that have been reported involve either chemical or enzymatic depolymerisation of GAGs followed by separation of non-derivatised (native) or derivatised disaccharide subunits and detection by either UV/fluorescence or MS. However, the measurement of these disaccharides is challenging due to their hydrophilic and labile nature. Here we report a pre-column LC-MS method for the quantification of GAG disaccharide subunits. Heparan sulphate (HS) was extracted from cell lines using a combination of molecular weight cutoff and anion exchange spin filters and digested using a mixture of heparinases I, II and III. The resulting subunits were derivatised with procainamide, separated using hydrophilic interaction liquid chromatography and detected using electrospray ionisation operated in positive ion mode. Eight HS disaccharides were separated and detected together with an internal standard. The limit of detection was found to be in the range 0.6–4.9 ng/mL. Analysis of HS extracted from all cell lines tested in this study revealed a significant variation in their composition with the most abundant disaccharide being the non-sulphated ∆UA–GlcNAc. Some structural functional relationships are discussed demonstrating the viability of the pre-column method for studying GAG biolog

The effect of irradiation and hyperbaric oxygenation (HBO) on extracellular matrix of the condylar cartilage after mandibular distraction osteogenesis in the rabbit.

The effects of irradiation and hyperbaric oxygenation (HBO) on the extracellular matrix of condylar cartilage after mandibular distraction were evaluated. Unilateral distraction was performed on 19 rabbits. Five study groups were included: control, low- and high-dose irradiation, and low- and high-dose irradiation groups with HBO. Additionally, four temporomandibular joints (TMJ) were used as control material. The high-dose irradiated animals were given in the TMJ 22.4 Gy/4 fractions irradiation (equivalent to 50 Gy/25 fractions). Low-dose irradiation group received a 2.2 Gy dosage. Two groups were also given preoperatively HBO 18 x 2.5ATA x 90 min. After a two-week distraction period (14 mm lengthening) and four-week consolidation period the TMJs were removed. Proteoglycan (PG) distribution of the extracellular matrix was evaluated using safranin O staining and collagen I and II using immunohistochemistry. The organization of fibrillar network was studied by polarized light microscopy. On the operated side of the control group and on the unoperated side in all, except for high-dose irradiated group, PG distribution and fibrillar network were normal appearing. In the irradiated groups, with or without HBO, the cartilaginous layer was partially or totally devoid of PG and the network structure was severely damaged. In conclusion, irradiation in conjunction with the pressure applied by distraction causes severe damage to extracellular matrix of condylar cartilage