From each paired sample, a ~2.5 mm x 2.5 mm corner section was cut for high performance liquid chromatography (HPLC). Bone segments were demineralized in 20% ethylenediaminetetraacetic acid (EDTA) at 4 °C. Then, these bone segments were hydrolyzed in 6 N HCl with 4.5 mM alpha-amino-N-butyric acid (α-ABA) for ~20 hours. After removal of the acid and filtration through 0.2 µm syringe filter, a portion of the re-suspended hydrolysate was used to measure hydroxyproline (to give mole of collagen) via the chromatogram generated by a UV detector (Beckman Coulter 168 Detector, Brea, CA) and following our previously published HPLC method (56 (link)).
To determine crosslink concentrations, another portion of the re-suspended hydrolysate was diluted in acetonitrile-heptafluorobutyric acid solution (10% v/v) with 0.25 µg/ml of pyridoxine (PYR) as the internal standard. Samples were injected along with standards consisting of PYR and varying concentrations of pyridinoline (PYD), deoxypyridinoline (DPD) and pentosidine (PEN) into a silica-based, reversed-phase C18 column (Waters Spherisorb® 5μm ODS2, Milford, MA)(57 (link)). A programmable fluorescence detector (Waters 2475 Multi λ Fluorescence Detector) was used to generate chromatograms. Moles of each crosslink per sample were divided by the corresponding moles of collagen.
To determine crosslink concentrations, another portion of the re-suspended hydrolysate was diluted in acetonitrile-heptafluorobutyric acid solution (10% v/v) with 0.25 µg/ml of pyridoxine (PYR) as the internal standard. Samples were injected along with standards consisting of PYR and varying concentrations of pyridinoline (PYD), deoxypyridinoline (DPD) and pentosidine (PEN) into a silica-based, reversed-phase C18 column (Waters Spherisorb® 5μm ODS2, Milford, MA)(57 (link)). A programmable fluorescence detector (Waters 2475 Multi λ Fluorescence Detector) was used to generate chromatograms. Moles of each crosslink per sample were divided by the corresponding moles of collagen.
