First, the lysine and hydroxylysine residues in colla-Pyridinoline (Pyr) and deoxypyridinoline (Dpyr) are gen molecules are deaminated by lysyl oxidase, and mature crosslinks which maintain the structure of the they nonenzymatically reacted with lysine and hycollagen fibril. Pentosidine (Pen) is a senescent crossdroxylysine in the next molecule of collagen. This link and one of the advanced glycation end products. formed crosslink is called a Schiff base, which is un-We developed a direct and one-injection method to stable and reducible by sodium borohydride. The measure Pyr, Dpyr, and Pen in the hydrolysate of tis-Schiff-base crosslinks undergo further unknown nonsues using reversed-phase high-performance liquid enzymatical rearrangement to form stable and nonchromatography. Using a linear gradient of acetonireducible crosslinks. Pyridinoline (Pyr), 1 a trifunctrile and a cleaning step, the objective crosslinks were tional 3-hydroxypyridinium crosslink (1), and its well separated and continuously and automatically asminor analogue, deoxypyridinoline (Dpyr) (2), are sayed. Recovery rates of Pyr, Dpyr, and Pen were 95two of those stable crosslinks. They are physiologi-116, 94-110, and 92-120%, respectively (n ร
5). The cally essential and necessary to maintain the strucintraassay coefficients of variation for Pyr, Dpyr, and ture of the collagen fibril network (3).
Pen were 5.3, 5.8, and 4.3%, respectively (n ร
5), and On the other hand, there is evidence that crosslinks the interassay coefficients of variation for Pyr, Dpyr, play a major role in collagen's changes with aging, reand Pen were 3.5, 4.6, and 5.7%, respectively (n ร
5).
sulting in loss of elasticity, decreased proteolytic sus-Linear regression analysis showed the linearity (r ร
ceptibility, and accumulation of yellow and fluorescent 0.999) of calibration line for each Pyr, Dpyr, and Pen.
substances. A glycation product can be this type of
We measured the content of these crosslinks in the crosslink. Chronic hyperglycemia leads to the accumutissues from the young and old subjects. There was no lation of nonenzymatically derived glycation products.
difference in the content of Pyr and Dpyr between the young and the old group. On the other hand, the con-These early, unstable glycation products undergo a tent of Pen in the old group was extremely higher than slow, time-consuming rearrangement to form irreversthat in the young group. We demonstrated the direct ible advanced glycation end products (AGE) (4). Almethod for measuring two kinds of major crosslinks though it is expected that there are many kinds of which have different characters and believe that this AGEs, only a few are known. Pentosidine (Pen), a conmethod will be useful in determining the content of densation product of arginine, lysine, and ribose, is one these crosslinks in tissues under various conditions. of those AGEs and is reported to increase with age and