Matrix Degradation Monitored as Urinary Levels of Collagen Crosslinks

Matrix Degradation Monitored as Urinary Levels of Collagen Crosslinks 

The HPLC equipment (Waters, Eschbom, Germany) consists of an isocratic pump no. 510, an injector no. 717, and a temperature control module. The pyridinoline crosslinks are separated on NovaPak C-18 (140 3.9 mm, 5 im particles) isocratic by 20 mM/1 heptafluorobutyric acid in 725 ml water/175 ml acetonitrile (1 ml/min) at 30 °C. Crosslinks urine standard containing 953 pM/ml PYR and 212 pM/ml dPYR (Biorad, Munich, Germany) was used for 

The experimental results are expressed as nM urinary pyridinium or desoxypyridinium per mM creatinine. 

This non-invasive method allows to follow an experiment longitudinally, and relates to more than one skeletal tissue. Care has to be taken however to not only normalize the values in respect to creatinine levels, in order to compensate for individual differences in urine concentration, but also to either collect the samples over a longer period than 24 h, as proposed by Smith et al. (2004), or to collect each time at the same hour to exclude the known circadian variation in excretion (Stone et al. 1998). In addition, when using female animals, the measurements should be recorded regularly over a longer period to correct for considerable peak variation during the estrous cycle (Blanque etal. 2001). 

Bank et al. (1997) developed a sensitive single-step HPLC procedure, by which hydrolysates from a wide variety of tissue sources can be analysed without prior sample treatment down to a detection limit of 0.4 pM for the PYR/dPYR crosslinks. 

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I.L.2.1 Matrix Degradation Monitored as Urinary Levels of Collagen Crosslinks 251... 

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