Quantitative Determination of Collagen Crosslinks - Methods in Molecular Biology: Extracellular Matrix Protocols - page 10

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Quantitative Determination of Collagen Crosslinks

Trevor J. Sims, Nicholas C. Avery, and Allen J. Bailey

1. Introduction
The primary functional role of collagen is as a supporting tissue and it is
now well established that the aggregated forms of the collagen monomers are
stabilized to provide mechanical strength by a series of intermolecular
crosslinks. These links are formed by oxidative deamination of the -amino

group of the single lysine in the amino and carboxy-telopeptides by lysyl oxi-
dase. The aldehyde thus formed reacts with an

ε

-amino group of a lysine at a
specific point in the triple helix because of the quarter-staggered end-overlap
alignment of the molecules in the fibers. The chemistry of these crosslinks is
dependent on both the nature and age of the collagenous tissue (1 , 2) . Differ-
ences in the crosslinks are because of the degree of hydroxylation of both the
telopeptide and the specific lysine in the triple helix. Thus, the amounts of
intermediate crosslinks present in immature tissue, dehydro-hydroxy-
lysinonorleucine (

ε

-HLNL), and hydroxylysino-keto-norleucine (HLKNL)
may vary considerably between tissues, e.g., rat tail tendon and skin contain
∆

∆

-HLNL whereas cartilage and bone contain predominantly HLKNL.
These divalent crosslinks are only intermediates and are subsequently con-
verted into stable trivalent crosslinks that accumulate in the tissue as collagen
turnover decreases during maturation (2) . The Schiff base aldimine -HLNL is

stabilized by reaction with histidine to form the trivalent crosslink, histidino-
hydroxlysinonorleucine (HHL). The keto-imine HLKNL, on the other hand,
reacts with a second hydroxylysyl aldehyde to form the pyridine derivatives,
hydroxylysyl-pyridinoline (Hyl-Pyr), and lysyl-pyridinoline (Lys-Pyr), Fig. 1 .
The proportion of these three known mature crosslinks, again varies with age
and with the type of tissue. For example, HHL is the major, known, mature
crosslink of human and bovine skin (3) , whereas the pyridinolines are the

∆