title: Simultaneous free and glycosylated pyridinium crosslink determination in urine: validation of an HPLC-fluorescence method using a deoxypyridinoline homologue as internal standard.
abstract: Pyridinoline (Pyr), deoxypyridinoline (D-Pyr), galactosyl-pyridinoline (Gal-Pyr) and glucosyl-galactosyl pyridinoline (GluGal-Pyr) are enzymatic mature pyridinium crosslinks. Generally, only total Pyr and D-Pyr urinary amounts (free+bound forms) are evaluated by HPLC as indices of bone resorption. This report describes the validation of an HPLC-fluorescence method for the simultaneous evaluation of free Pyr and D-Pyr, together with GluGal-Pyr and Gal-Pyr, in urine of healthy women (n=20, aged 27-41) and girls (n=20, aged 5-10). The use of an unnatural D-Pyr homologue, here proposed for the first time as internal standard, and of pure Pyr, D-Pyr, GluGal-Pyr and Gal-Pyr synthesized to be used as primary calibrators, guarantees method specificity and correct crosslink quantification. Urine, spiked with IS, was solid-phase extracted prior to HPLC analysis. Total Pyr and D-Pyr amounts were also evaluated after urine hydrolysis. The HPLC method was validated for selectivity, sensitivity, linearity, precision, accuracy, recovery and stability for all measured crosslinks. Both free and total Pyr and D-Pyr as well as GluGal-Pyr and Gal-Pyr amounts were significantly higher in girls than in women (p<0.0001), indicating an increased collagen turnover rather than only bone turnover. Gal-Pyr, for the first time evaluated in girls, was under its lower quantification limit (<LLOQ, <21.20 pmol/mL) in women. The measurement of free and glycosylated pyridinium crosslinks might provide more information on the degradation of various types of collagen than only that of total Pyr and D-Pyr. Moreover, this validated method could be a useful non-invasive technique for studying pathological conditions characterized by modified glycosylation enzyme activity and for more clinical investigation on bone fragility.
authors.affiliation: Dipartimento di Scienze Cliniche Luigi Sacco, Università degli Studi di Milano, Via G.B. Grassi 74, 20157 Milan, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Monticelli
authors.forename: Caroline Stéphanie
authors.initials: CS
authors.lastname: Aman
authors.forename: Maria Letizia
authors.initials: ML
authors.lastname: Costa
authors.forename: Paola
authors.initials: P
authors.lastname: Rota
authors.forename: Daniela
authors.initials: D
authors.lastname: Bogdan
authors.forename: Pietro
authors.initials: P
authors.lastname: Allevi
authors.forename: Giuliana
authors.initials: G
authors.lastname: Cighetti
mesh_terms: D000328:Adult; D000596:Amino Acids / Q000032:analysis / Q000737:chemistry / Q000652:urine*; D001862:Bone Resorption / Q000652:urine; D002648:Child; D002675:Child, Preschool; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004355:Drug Stability; D005260:Female; D005697:Galactosides / Q000737:chemistry / Q000652:urine*; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D016018:Least-Squares Analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
chemical_list: D000596:Amino Acids; D005697:Galactosides; C015484:pyridinoline; C039434:galactosylpyridinoline; C036020:deoxypyridinoline