A PROPOSED NEW INTERNAL STANDARD FOR FREE, GLYCOSYLATED AND TOTAL PYRIDINIUM CROSSLINKS QUANTIFICATION IN HEALTHY WOMEN AND CHILDREN URINE: VALIDATION OF AN HPLC-FLUORESCENCE METHOD
Creators: name: MONTICELLI, ELENA
Publisher: name: Universit� degli Studi di Milano
Year: 2010

description: Emerging evidence supports the concept that biochemical markers are clinically useful as non invasive diagnostic tools for the monitoring of changes in bone and cartilage turnover in subjects affected by pathologies with destructive bone and joint diseases such as osteoporosis, osteoarthritis, rheumatoid arthritis and osteosarcoma. Epidemiological studies demonstrated that the measurements of different bone degradation products in urine or serum samples are higher in patients affected by the above reported pathologies compared with healthy subjects. Among the different biochemical markers used for clinical purpose, the changing in urine concentrations of pyridinium crosslinks is usually measured. Pyridinium crosslinks are generally referred to the chemical compounds pyridinoline (Pyr) and deoxypyridinoline (D-Pyr), both considered specific markers of skeletal collagen degradation. They are released into the circulation during bone resorption as small peptides that can be further degradated into free crosslinks in kidney and excreted in urine in both free and peptides or sugars bound forms as the disaccharide glucose-galactose Pyr (GluGal-Pyr) and the monosaccharide galactose Pyr (Gal-Pyr), both formed from a post-translation modification of collagen. The first sugar bound form, reported absent in bone by some authors, is considered an important predictor of joint tissue degradation, whereas for the second one, mainly present in bone, no specific role has been found and it has never been measured. Commonly, only the total Pyr and D-Pyr (free+bound forms) amounts, obtained from urine submitted to overnight acid hydrolysis, are measured as markers of bone resorption by high-performance liquid chromatography (HPLC) coupled to a fluorescence detector. However, the variability of literature results, depending on analytical problems (hydrolysis, solide-phase purification, recovery) and physiological factors (age, gender) renders the results from different analytical laboratories difficult to be compared. Moreover, results obtained from different analytical methods are often compromised by the lack of an adequate internal standard allowing to eliminate errors occurring during sample preparation and analytical measurement. In spite of extensive literature, important points for crosslinks measurement have not been solved: 1) the choice of an adequate internal standard, required by many researchers working in this field, that allows the specificity of the analytical method and avoids errors occurring during all the pre-analytical steps; 2) the pure authentic reference standards, obtained by synthesis, to be used as primary calibrators necessary to improve the crosslinks quantification process; 3) the evaluation of crosslinks in their total (free + bound forms) and/or free forms. In fact the assessment of total urinary D-Pyr, found only in bone and dentine, provides an index of bone resorption whereas the evaluation of total Pyr does not allow to distinguish among the degradation of bone, synovium and joint and between free and glycosylated-Pyr. The aim of the present study was to develop and validate an HPLC-fluorescence method for the measurement of Pyr and D-Pyr in free, glycosylated and total forms in healthy women and in children urine. Women were selected to compare our results with those reported by others in adult healthy groups. Children were enrolled as, being in growing age and having an extremely high remodelling rate, their physiological levels of urine collagen crosslinks in this stage of the life appeared of particular interest to be compared with those of an omogeneous group of pre-menopauseal women. Moreover, studies on children were performed in order to estimate GluGal-Pyr and Gal-Pyr amounts as these glycosylated pyridinolines have never been measured until now. The specificity of the method was assured by a synthesized superior homologue of D-Pyr, here proposed as internal standard (IS) for the first time, added to the biological samples before any pre-analytical step. Moreover, pure synthesized Pyr, D-Pyr, Gal-Pyr and GluGal-Pyr were used as primary calibrators for the correct HPLC identification of each analyte by their corresponding retention times and for their accurate quantification by specific calibration curves. To validate the method for the measurement of all the crosslinks of interest, various analytical performance parameters (linearity, recovery, within and between-day repeatabilities, reproducibility, limit of detection, limit of quantitation) have been considered and here reported. The results demonstrate, for the validated method, good linearity, recovery, precision, limit of detection and limit of quantitation for all compounds. The measurement of consisting amounts of GluGal-Pyr in both women and children urine and few but detectable Gal-Pyr amounts only in children confirm the importance for free and glycosylated crosslinks quantification. In fact, only the evaluation of free glycosylated and total crosslinks excretion can provide more information on different collagen catabolism, since total crosslinks amounts give no information on the intermediate products (pyridinoline glycosides) generated by various collagen degradations.