Abstract
Reactive oxygen species produced by activated neutrophils and monocytes are thought to be involved in mediating the loss of collagen and other matrix proteins at sites of inflammation. To evaluate their potential to oxidize the pyridinoline (Pyd) cross-links found in collagen types I and II, we reacted hydrogen peroxide (H(2)O(2)), hypochlorous acid/hypochlorite (HOCl/OCl(-)), and singlet oxygen (O(2)((1)delta g)) with the Pyd substitutes, pyridoxamine dihydrochloride and vitamin B(6), which share the same chemical structure and spectral properties of Pyd cross-links. Neither H(2)O(2) (125-500 microm) nor O(2)((1)delta g) (10-25 microm) significantly changed the spectral properties of pyridoxamine or vitamin B(6). Reaction of HOCl/OCl(-) (12.5-50 microm) with pyridoxamine at pH 7.2 resulted in a concentration-dependent appearance of two new absorbance peaks and a decrease in fluorescence at 400 nm (excitation 325 nm). The new absorbance peaks correlated with the formation of an N-chloramine and the product of its subsequent reaction with pyridoxamine. In contrast, the extent to which HOCl reacted with vitamin B(6), which lacks a primary amine group, was variable at this pH. At lysosomal pH 5.5, Cl(2)/HOCl/OCl(-) reacted with both pyridoxamine and vitamin B(6). Four of the chlorinated products of this reaction were identified by gas chromatography-mass spectrometry and included 3-chloropyridinium, an aldehyde, and several chlorinated products with disrupted rings. To evaluate the effects of Cl(2)/HOCl/OCl(-) on Pyd cross-links in collagen, we exposed bone collagen type I and articular cartilage type II to HOCl. Treatment of either collagen type with HOCl at pH 5. 0 or 7.2 resulted in the oxidation of amine groups and, for collagen type II, the specific decrease in Pyd cross-link fluorescence, suggesting that during inflammation both oxidations may be used by neutrophils and monocytes to promote the loss of matrix integrity.
Highlights
Reactive oxygen species produced by activated neutrophils and monocytes are thought to be involved in mediating the loss of collagen and other matrix proteins at sites of inflammation
Oxidative Modification of Pyridoxamine and Vitamin B6 after Exposure to the reactive oxygen species (ROS), H2O2, hypochlorous acid (HOCl)/OClϪ, or O2(1⌬g)—To evaluate the oxidation of pyridoxamine or vitamin B6 by H2O2, HOCl/OClϪ, or O2(1⌬g), a 15 M solution of pyridoxamine or vitamin B6 dissolved in 0.5 M sodium phosphate buffer, pH 7.2 Ϯ 0.2, was exposed for 15 min at RT to 125, 250, or 500 M of H2O2, 12.5, 25, or 50 M of HOCl, or a combination of H2O2/HOCl in a ratio of 10:1
The above concentrations of HOCl and H2O2 are within the predicted range generated by activated neutrophils or monocytes at sites of inflammation [6]
Summary
POSSIBLE ROLE OF HYPOCHLORITE, N-CHLORAMINES, AND CHLORINE IN THE OXIDATION OF PYRIDINOLINE CROSS-LINKS OF ARTICULAR CARTILAGE COLLAGEN TYPE II DURING ACUTE INFLAMMATION*. Inflammation associated with articular cartilage, bone, and dentin surfaces is characterized by accumulation, adhesion, and activation of neutrophils and monocytes, which results in the destruction of cartilage and the loss of bone at these sites [1,2,3,4] These effects are thought to be mediated in part by the production of reactive oxygen species (ROS), a group of reactants that includes superoxide (O2.), hydrogen peroxide (H2O2), and the highly reactive species hypochlorous acid (HOCl) [5,6,7,8]. Our findings indicate that these cross-links react with OClϪ, Cl2, and N-chloramines and suggest that Pyd would be a site for ROS modification of collagen type I and II in bone and cartilage, respectively
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