Direct Detection of Crosslinks of Collagen and Elastin in the Hydrolysates of Human Yellow Ligament Using Single-Column High Performance Liquid Chromatography

Abstract

Collagen and elastin are recognized as two major connective tissue proteins of human yellow ligament. In both collagen and elastin there are many kinds of intra- or intermolecular crosslinks. Pyridinoline (Pyr) and deoxypyridinoline (Dpyr) are mature crosslinks which maintain the structure of the collagen fibril. Desmosine (Des) and isodesmosine (Isodes) represent the major crosslinking components of elastin. Pentosidine (Pen), which is a senescent crosslink and one of the advanced glycation end products, accumulates with age in tissue proteins including collagen. We developed a direct and one-injection HPLC method to measure Pyr, Dpyr, Des, Isodes, and Pen in the hydrolysate of human yellow ligament. This method used one column and two detectors. Recovery rates of Pyr, Dpyr, Pen, Des, and Isodes were 86.4–98.3, 83.6–96.8, 78.7–95.6, 83.6–97.9, and 85.6–99.3%, respectively (n = 8). The intraassay coefficients of variation for Pyr, Dpyr, Pen, Des, and Isodes were 3.7, 4.1, 5.4, 4.5, and 4.7%, respectively (n = 8), and the interassay coefficients of variation for Pyr, Dpyr, Pen, Des, and Isodes were 4.4, 5.1, 4.9, 4.6 and 4.1%, respectively. Linear regression analysis showed the linearity (r = 0.99, P = 0.0001) of calibration line for each Pyr, Dpyr, Pen, Des, and Isodes. Using this method, we investigated age-related changes in the crosslinks of collagen and elastin in human yellow ligament. There was a significant correlation between Pen and age, but no correlations with Pyr, Dpyr, Des, and Isodes. We believe that this method is useful for investigating the content of these crosslinks in both collagen and elastin under various conditions.