Standardization of pyridinium crosslinks, pyridinoline and deoxypyridinoline, for use as biochemical markers of collagen degradation

Abstract

The collagen crosslinks, pyridinoline and deoxypyridinoline, have been developed as urinary markers of bone resorption but, despite wide clinical application of the technique, comparatively little attention has been paid to the standardization of these compounds. In this study, pyridinoline and deoxypyridinoline have been purified from bone and converted completely to monochloride trihydrochloride salts. In addition to mass spectrometry and NMR spectroscopy, the purity of the isolated materials was assessed by microelemental analysis including the chloride counterions. These purified compounds were used to establish individual molar absorptivity values as primary standardization criteria for the two crosslinks. For pyridinoline in 0.1 mol/L HCl, epsilon at 295 nm was 5490 L mol(-1) cm(-1); in 50 mmol/L sodium phosphate, pH 7.5, epsilon at 325 nm was 5785. The corresponding values for deoxypyridinoline at acid and neutral pH were 5160 and 5290 L mol(-1) cm(-1). The availability of standardization criteria for the crosslinks will allow more meaningful comparisons of clinical data between different laboratories.